Methods for treating resistant diseases using triazole containing macrolides

ABSTRACT

Described herein are macrolide and ketolide antibiotics and pharmaceutical compositions, methods, and uses thereof for treating diseases caused at least in party by resistant bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Application Ser. No. 61/108,110, filed on Oct. 24, 2008,U.S. Provisional Application Ser. No. 61/108,112, filed on Oct. 24,2008, U.S. Provisional Application Ser. No. 61/108,134, filed on Oct.24, 2008, U.S. Provisional Application Ser. No. 61/108,137, filed onOct. 24, 2008, U.S. Provisional Application Ser. No. 61/108,168, filedon Oct. 24, 2008, and U.S. Provisional Application Ser. No. 61/162,109,filed on Mar. 20, 2009, the entire disclosure of each of which areincorporated herein by reference.

TECHNICAL FIELD

The invention described herein relates to the treatment of resistantdiseases, or more particularly, diseases caused at least in part by oneor more resistant organisms. In particular, the invention describedherein relates to the treatment of resistant diseases, or moreparticularly, diseases caused at least in part by one or more resistantorganisms with triazole-containing macrolide and ketolide antibiotics.

BACKGROUND AND SUMMARY OF THE INVENTION

Since the discovery of erythromycin A (ERY), macrolide antibiotics(macrolides) have been an important class of molecules for treating awide variety of bacterial infections. Further, research on macrolideshas provided the discovery of several new generations of macrolideantibiotics. One group is the clarithromycin (CLR) class of compounds,where the ring structure was stabilized by the methylation of the C-6hydroxyl. Another group is the 15-membered ring aza analogs, of whichazithromycin (AZI) is a notorious member. Another groups is the3-desglycosyl-3-oxo analogs, also known as ketolides, of whichtelithromycin (TEL) and cethromycin (CTH) are notorious members.However, as has been true for other antibiotic classes of molecules,such as the penecillins, cephalosporins, quinolones, vancomycin, andothers, resistant organisms have emerged throughout the world. Macrolideresistance, which is now predominant in some countries such as Japan andKorea, has been blamed on the overuse of AZI and CLR during the past 15years. It has also been observed that macrolide resistance also usuallyoccurs together with penicillin G resistance (though geneticallyunlinked). For example, though all strains of group A streptococci arestill β-lactam susceptible, macrolide resistance has occurred,especially in Asia and in southern, central and eastern Europe.Infections caused by drug-resistant group A streptococci are encounteredworldwide and sometimes life-threatening infections caused by theseorganisms are encountered. Further, Streptococcus pyogenes strains,although retaining their β-lactam susceptibility are becoming moremacrolide resistant. It is estimated that nearly 40% of Streptococcusstrains in the US are resistant to both penicillin and macrolideantibiotics.

In fact, it has been suggested that the introduction of TEL into thetherapeutic armamentarium was intended to solve the problem of macrolideresistance in streptococci. TEL is effective against penicillin anderythromycin-resistant S. pneumoniae and is a non-inducer ofMacrolide-Lincosamide-Streptogramin B (MLS_(B)) resistance. However,even with TEL, which is active against many macrolide resistant S.pyogenes genotypes, resistant species have and continue to emerge. Inparticular, ketolide resistant species, namely to TEL, but also possiblycross resistant with CTH, have been reported worldwide, and mostrecently in S. pyogenes from Europe. Further, TEL is not active againsterm(B) group A streptococci (which are naturally TEL resistant).Moreover, observed TEL toxicities have limited the clinical utility ofthis drug. The rapid emergence of resistant strains may not besurprising; when the free AUC/MIC of TEL against macrolide-resistantpneumococci even with low MICs is examined carefully, it is observedthat the number was not significantly above 25. Thus, the highprobability of resistance developing might be predicted to occur.

In addition, though the pediatric conjugate vaccine has dramaticallydecreased meningitis and bacteremia caused by most of the usualdrug-resistant pneumococcal clones, outbreaks of serious cases of otitismedia caused by pan-resistant strains with a serotype (19A) not includedin the vaccine have been reported. Thus, the problem of drug-resistantpneumococci causing community-acquired respiratory infection, especiallyin children, is likely to worsen with the spread of this clone.

Several specific macrolide resistant mechanisms have been reported,including ribosomal methylation-based resistance (erm(A), erm(B)),efflux-based resistance (mef(A), mef(E), mef(I)), and resistance arisingfrom mutation of the rRNA or ribosomal protein, such as 23S and L4mutations.

Accordingly, a continuing need for new antibiotics and anti-bacterialagents remains. Further, those new agents would desirably have theproperty of a low potential for resistance development or induction, anda low potential for naturally occurring resistance.

It has been surprisingly discovered herein that triazole-containingmacrolides, including ketolides, exhibit high activity in vitro and invivo against numerous organisms. Moreover, it has been discovered thatthe triazole-containing macrolides described herein exhibit highactivity in vitro and in vivo against numerous resistant organisms,including both macrolide and ketolide resistant organisms.

In one illustrative embodiment, compounds of Formula (I) are describedherein

including pharmaceutically acceptable salts, hydrates, solvates, esters,and prodrugs thereof.

In one aspect, R₁₀ is hydrogen or acyl. In another aspect, X is H; and Yis OR₇; where R₇ is a monosaccharide or disaccharide, alkyl, aryl,heteroaryl, acyl, or C(O)NR₈R₉, where R₈ and R₉ are each independentlyselected from the group consisting of hydrogen, hydroxy, alkyl, aralkyl,alkylaryl, heteroalkyl, aryl, heteroaryl, alkoxy, dimethylaminoalkyl,acyl, sulfonyl, ureido, and carbamoyl; or X and Y are taken togetherwith the attached carbon to form carbonyl.

In another aspect, V is C(O), C(═NR₁₁), CH(NR₁₂, R₁₃), or N(R₁₄)CH₂,where N(R₁₄) is attached to the C-10 carbon of the compounds of FormulaeI and 2; wherein R₁₁ is hydroxy or alkoxy, R₁₂ and R₁₃ are eachindependently selected from the group consisting of hydrogen, hydroxy,alkyl, aralkyl, alkylaryl, alkoxy, heteroalkyl, aryl, heteroaryl,dimethylaminoalkyl, acyl, sulfonyl, ureido, and carbamoyl; R₁₄ ishydrogen, hydroxy, alkyl, aralkyl, alkylaryl, alkoxy, heteroalkyl, aryl,heteroaryl, dimethylaminoalkyl, acyl, sulfonyl, ureido, or carbamoyl.

In another aspect, W is H, F, Cl, Br, I, or OH.

In another aspect, A is CH₂, C(O), C(O)O, C(O)NH, S(O)₂, S(O)₂NH,C(O)NHS(O)₂. In another aspect, B is (CH₂)_(n) where n is an integerranging from 0-10, or B is an unsaturated carbon chain of 2-10 carbons.In another aspect, C is hydrogen, hydroxy, alkyl, aralkyl, alkyl aryl,alkoxy, heteroalkyl, aryl, heteroaryl, aminoaryl, alkylaminoaryl, acyl,acyloxy, sulfonyl, ureido, or carbamoyl.

In another embodiment, compositions including a therapeuticallyeffective amount of one or more compounds of formula (I), or the varioussubgenera thereof are described herein. The pharmaceutical compositionsmay include additional pharmaceutically acceptable carriers, diluents,and/or excipients.

In another embodiment, methods are described herein for treatingdiseases arising from pathogenic organism populations. The methodsinclude the step of administering a therapeutically effective amount ofone or more compounds of formula (I), or the various subgenera thereofare described herein, to a patient in need of relief or suffering from adisease caused by a pathogenic organism.

In another embodiment, uses are described herein for the manufacture ofmedicaments. The medicaments include a therapeutically effective amountof one or more compounds of formula (I), or the various subgenerathereof are described herein, or one or more compositions thereofdescribed herein. The medicaments are suitable for treating diseasesarising from pathogenic organism populations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparative susceptibilities of S. aureus ATCC 25923 and L.monocytogenes EGD to CEM-101, TEL, AZI, and CLR, based on MICdeterminations in pH-adjusted broth.

FIG. 2. Short-term time-kill effect of CEM-101 and AZI on S. aureus(ATCC 25923) in broth (left panels; pH 7.4) or after phagocytosis byTHP-1 macrophages (right panels). Both drugs were used at anextracellular concentration of either 0.7 (top panels) or 4 (bottompanels) mg/liter. MICs of CEM-101 and AZI were 0.06 and 0.5 mg/liter,respectively. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 3. Concentration-effect relationships for CEM-101, TEL, CLR, andAZI toward S. aureus (ATCC 25923) in broth (left panels) and afterphagocytosis by THP-1 macrophages (right panels). The ordinate shows thechange in CFU (A log CFU) per ml (broth) or per mg of cell protein(THP-1 macrophages) at 24 h compared to the initial inoculum. Theabscissa shows the concentrations of the antibiotics as follows: (i) toppanels, weight concentrations (in mg/liter) in broth (left) or in theculture medium (right) and (ii) bottom panels, multiples of the MIC asdetermined in broth at pH 7.4. All values are means±standard deviations(SD) of three independent experiments (when not visible, SD bars aresmaller than the symbols). Statistical analysis based on global analysisof curve-fitting parameters (one-way analysis of variance); the onlysignificant difference is between CEM-101 and AZI in broth (P=0.04).Numerical values of the pertinent pharmacological descriptors andstatistical analysis of their differences are shown in Table 1.

FIG. 4. Concentration-effect relationships for CEM-101 and AZI towardintraphagocytic L. monocytogenes (strain EGD, left panels) and L.pneumophila (strain ATCC 33153, right panels). The ordinate shows thechange in CFU (Δ log CFU) per mg of cell protein at 24 h (L.monocytogenes) or 48 h (L. pneumophila) compared to the initialpostphagocytosis inoculum. The abscissa shows the concentrations of theantibiotics as follows: (i) top panels, weight concentrations (inmg/liter); (ii) bottom panels, multiples of the MIC as determined inbroth at pH 7.4. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 5. Accumulation of CEM-101 versus comparators in THP-1 cells at 37°C. (all drugs at an extracellular concentration of 10 mg/liter). (A)Kinetics of accumulation (AZI); Cc, intracellular concentration; Ce,extracellular concentration); (B) influence of the pH of the culturemedium on the accumulation (30 min) of CEM-101 (solid symbols and solidline) and AZI (open symbols and dotted line); (C) influence of monensin(50 μM; 2-h incubation), verapamil (150 μM; 24-h incubation), orgemfibrozil (250 μM; 24-h incubation) on the cellular accumulation ofAZI and CEM-101. All values are means±standard deviations (SD) of threeindependent determinations (when not visible, SD bars are smaller thanthe symbols).

FIG. 6. Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages. Bars represent the MICs (in mg/L) or theextracellular static dose.

FIG. 7. Intracellular Activity of CEM-101 compared to AZI, CLR, and TEL,expressed as a dose response curve of Δ log CFU from time 0 to 24 hoursversus log dose.

DETAILED DESCRIPTION

The compounds, compositions, methods, and medicaments described hereinincluding triazole-containing macrolides and ketolides are useful intreating various diseases caused by pathogenic organism populations.Such pathogenic organisms are well known to cause a variety of diseasesand disease states. It is appreciated that in some cases the disease ordisease state may be characterizable as a symptom of some otherunderlying disease. In such cases, it is to be understood that suchsymptom or symptoms are a disease treatable using the compounds,compositions, methods, and medicaments described herein.

In one embodiment, the compounds, compositions, methods, and medicamentsdescribed herein are useful for treating respiratory tract infections(RTIs), including community acquired RTIs, such as those arising from orcomplicated by susceptible bacterial pathogens and/or MLS_(B) resistantpathogens.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byH. influenzae, such as lower respiratory tract infections, bacteremia,pneumonia, otitis media, conjunctivitis, sinusitis and acute bacterialmeningitis, such as may occur in infants and young children, and causedmore specifically by H. influenzae type b (Hib). In another embodiment,the diseases include cellulitis, osteomyelitis, epiglottitis, and jointinfections.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by Mycoplasma, including M. pneumoniae, M. hominis, M.genitalium, and the like. Both Mycoplasma, including M. pneumoniae, M.hominis, M. genitalium, and M. fermentans, and Ureaplasma, including U.parvum and U. urealyticum can be responsible for infections in therespiratory and urogenital tracts. Illustrative diseases that may betreated using the compounds, compositions, methods, and medicamentsdescribed herein, that may be caused by mycoplasma include, but are notlimited to, respiratory disorders, including atypical pneumonia, humanprimary atypical pneumonia (PAP), walking pneumonia, and the like, suchas may be caused by M. pneumoniae, pelvic inflammatory diseases, such asmay be caused by M. genitalium, contagious bovine pleuropneumonia(CBPP), such as may be caused by M. mycoides, or subspecies of mycoidesSC (small-colony type), and the like. Illustrative diseases that may betreated using the compounds, compositions, methods, and medicamentsdescribed herein, that may be caused by ureaplasma include, but are notlimited to, aminolysis disorders, premature delivery disorders, and thelike. M. pneumoniae is a very small bacterium in the class Mollicutes,and is known to cause Mycoplasma pneumonia, a form of bacterialpneumonia. M. hominis is a strain of bacteria present in the vagina, andis believed to be a cause of pelvic inflammatory disease. M. hominis isknown to frequently colonize the genital tract of sexually active menand women. This bacterium has also been associated with post-abortal andpost-partum fever. M. genitalium was originally isolated in 1980 fromurethral specimens of two male patients with non-gonococcal urethritis.Infection by M. genitalium is fairly common and can be transmittedbetween partners during unprotected sexual intercourse.

In another embodiment, the compositions, methods, and medicamentsdescribed herein in a therapeutically amount of one or more compoundsdescribed herein, where the therapeutically effective amount is capableof exhibiting bactericidal activity against one or more mycoplasmaand/or ureaplasma organisms. In another embodiment, the compounds,compositions, methods, and medicaments described herein are useful fortreating diseases caused by or complicated by Mycoplasma that areresistant to macrolides, including ketolides. In another embodiment, thecompounds, compositions, methods, and medicaments described herein areuseful for treating diseases caused by or complicated by Mycoplasma thatare resistant to TEL and/or CTH. In another embodiment, the compounds,compositions, methods, and medicaments described herein are useful fortreating diseases caused by or complicated by Mycoplasma that areresistant to CLR. In another embodiment, the compounds, compositions,methods, and medicaments described herein are useful for treatingdiseases caused by or complicated by Mycoplasma that are resistant todoxycycline.

It is appreciated that macrolides reportedly have been the treatments ofchoice for M. pneumoniae respiratory infections of adults and childrenbecause they have the advantages of being safe and well tolerated inoral formulations, possess anti-inflammatory properties independent oftheir antibacterial activities, and activity against othermicroorganisms that may cause clinically similar illness. Theseproperties have also made macrolides attractive for empiric treatmentsince most mycoplasmal infections are never confirmed by microbiologicaltesting. However, many macrolides lack activity against M. fermentansand M. hominis. Moreover, it has been suggested that the likelihood thatmacrolide resistance will develop naturally in M. pneumoniae isplausible since there is only a single rRNA operon in the genome and invitro selected point mutations in domain V of 23S rRNA reduce theiraffinity for ribosomes.

For example, recent publications have confirmed the emergence ofmacrolide resistance in 10-33% of M. pneumoniae isolates that may haveclinical implications on patient outcome. It has been reported thatthose isolates typically have mutations in domain V of 23S rRNA anderythromycin MICs of 32->64 μg/ml. A recent report from Shanghai, Chinadescribed 39/50 (78%) of M. pneumoniae were macrolide-resistant. The USCenters for Disease Control and Prevention described 3 of 11 cases (27%)of M. pneumoniae infections from a recent outbreak that weremacrolide-resistant and had a 23S rRNA mutation. In addition, it hasbeen reported that two pediatric patients in Birmingham, Ala. withmacrolide-resistant M. pneumoniae infections of the lower respiratorytract did not respond initially to treatment with AZI and requiredseveral days of hospitalization (Xiao et al., Emerging macrolideresistance in Mycoplasma pneumoniae in children: detection andcharacterization of resistant isolates. Pediatr Infect Dis J In Press(2009)). Fluoroquinolone resistance has been described in genitalmycoplasmas (Bebear et al., In vitro activity of trovafloxacin comparedto those of five antimicrobials against mycoplasmas including Mycoplasmahominis and Ureaplasma urealyticum fluoroquinolone-resistant isolatesthat have been genetically characterized. Antimicrob Agents Chemother44:2557-60 (2000); Duffy et al., Fluoroquinolone resistance inUreaplasma parvum in the United States. J Clin Microbiol 44:1590-1(2006)) and tetracycline resistance may now exceed 40% in somepopulations (Waites et al., Mycoplasmas and ureaplasmas as neonatalpathogens. Clin Microbiol Rev 18:757-89 (2005)). AZI resistanceassociated with clinical treatment failure has also been documented inM. genitalium (Jensen et al., AZI treatment failure in Mycoplasmagenitalium-positive patients with nongonococcal urethritis is associatedwith induced macrolide resistance. Clin Infect Dis 47:1546-53 (2008)).Despite these challenges, it has been discovered that thetriazole-containing macrolides and ketolides described herein are usefulin treating diseases caused at least in part by Mycoplasmas and/orUreaplasmas, such as urethritis and other infections of the urethra andurogenital tract, including Mycoplasmas and/or Ureaplasmas resistant toother anti-bacterial agents, macrolides, and ketolides, such as TEL andCTH.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by Legionella sp., such as L. pneumophila, and the like.L. pneumophila is known to cause legionellosis, also known asLegionnaires' disease.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by staphylococcus and/or streptococci organisms, such asskin and skin structure infections (SSSIs). In one variation, the SSSIis a complicated SSSI. In another variation, the SSSI is anuncomplicated SSSI. Illustrative diseases caused by or complicated by S.aureus include, but are not limited to, minor skin infections, such aspimples, impetigo, boils, cellulitis, folliculitis, furuncles,carbuncles, scalded skin syndrome and abscesses, and more serious oreven life-threatening diseases such as pneumonia, meningitis,osteomyelitis, endocarditis, toxic shock syndrome (TSS), and septicemia.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by gonococci. Symptoms of infection with N. gonorrhoeaediffer depending on the site of infection. Illustrative diseases causedby or complicated by gonococci include both gonococcal andnon-gonococcal urethritis, which can result in a purulent (or pus-like)discharge from the genitals, inflammation, redness, swelling, dysuria,and/or a burning sensation during urination. Infection of the genitalsin females with N. gonorrhoeae can result in pelvic inflammatorydisease, and if left untreated, may result in infertility. Pelvicinflammatory disease may result if untreated N. gonorrhoeae travels intothe pelvic peritoneum (via the cervix, endometrium and fallopian tubes).

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by one or more Neisseria, such as N. meningitides,including organisms that are resistant nalidixic acid. It is appreciatedherein that resistance to nalidixic acid may correlate to resistance tofluoroquinolones. Accordingly, in another embodiment, the compounds,compositions, methods, and medicaments described herein are useful fortreating diseases caused by or complicated by one or more Neisseria,such as N. meningitides, that are resistant to fluoroquinolones, such astrimethoprim/sulfamethoxazole. N. meningitides infections may occur inthe nasopharynx.

N. gonorrhoeae can also cause conjunctivitis, pharyngitis, proctitis orurethritis, prostatitis and orchitis when present in other tissues. Forexample, conjunctivitis is reportedly common in neonates; accordinglysilver nitrate or antibiotics are often applied to newborns eyes as apreventive measure against gonorrhoea. Neonatal gonorrhealconjunctivitis is contracted when the infant is exposed to N.gonorrhoeae in the birth canal, and can result in corneal scarring orperforation. Disseminated N. gonorrhoeae infections can also occur,resulting in endocarditis, meningitis, and/or gonococcaldermatitis-arthritis syndrome. Dermatitis-arthritis syndrome is oftenaccompanied by arthralgia, tenosynovitis, and/or painless non-pruriticdermatitis.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by Enterococcus spp., including E. faecalis. Enterococcusspp., cause or contribute to diseases such as urinary tract infections,bacteremia, bacterial endocarditis, diverticulitis, and meningitis.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by ureaplasma sp, such as U. parvum, U. urealyticum, andthe like. U. urealyticum has been reportedly associated with a number ofdiseases in humans, including non-specific urethritis (NSU),infertility, chorioamnionitis, stillbirth, premature birth, and, in theperinatal period, pneumonia, bronchopulmonary dysplasia, and meningitis.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by Chlamydia sp., including C. trachomatis, C.pneumoniae, and the like. Like gonococci, Chlamydia causes differentdiseases depending upon the organ or tissue infected. Chlamydia sp.reportedly cause genital disease. For example, C. pneumoniae is wellrecognized as an important pathogen of respiratory tract infectionsworldwide, being responsible for almost 10% of cases ofcommunity-acquired pneumonia. Chlamydial infection of the neck of thewomb (cervicitis) is a sexually transmitted illness which isasymptomatic for about 50-70% of women infected with the disease.However, the infection can be passed through vaginal, anal, and/or oralsex. Even when asymptomatic, chlamydial infection that is not detectedwill progress to pelvic inflammatory disease (PID), in approximatelyhalf of affected persons. PID is a generic term for infection of theuterus, fallopian tubes, and/or ovaries. PID can cause scarring insidethe reproductive organs, later causing more serious complications,including chronic pelvic pain, difficulty becoming pregnant, ectopic(tubal) pregnancy, and other dangerous complications of pregnancy. Inaddition, it has been reported that women infected with chlamydia are upto five times more likely to become infected with HIV, if exposed. Inmen, Chlamydia reportedly shows symptoms of infectious urethritis(inflammation of the urethra) in about 50% of cases. If left untreated,it is possible for Chlamydia in men to spread to the testicles causingepididymitis, which in rare cases can cause sterility if not treated.Chlamydia has also been suggested as a potential cause of prostatitis inmen.

Chlamydia also reportedly causes eye disease, and in particularconjunctivitis due to chlamydia infection. Chlamydia conjunctivitis ortrachoma was once the most important cause of blindness worldwide, butits role is reportedly diminished. Newborns can also develop chlamydiaeye infection through childbirth via exposure to the organism in thebirth canal. Chlamydia also reportedly causes rheumatologicalconditions, such as reactive arthritis, or the triad of arthritis,conjunctivitis, and urethritis, especially in young men. Chlamydia alsoreportedly causes perinatal infections. It has been reported that asmany as half of all infants born to mothers with chlamydia will be bornwith the disease. Chlamydia can affect infants by causing spontaneousabortion; premature birth; conjunctivitis, which may lead to blindness;and pneumonia. Chlamydia also reportedly causes other conditions, suchas lymphogranuloma venereum, an infection of the lymph nodes andlymphatics, caused by C. trachomatis. Lymphogranuloma venereum usuallyis evidenced by genital ulceration and swollen lymph nodes in the groin,but it may also manifest as proctitis (inflammation of the rectum),fever or swollen lymph nodes in other regions of the body.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases caused byor complicated by Steptococci sp., such as S. pneumoniae, S. pyogenes,alpha-hemolytic streptococci, Streptococcus Viridans-group, thebeta-hemolytic streptococci of Lancefield groups A and B (also known as“Group A Strep” and “Group B Strep”), and the like. In addition to strepthroat, certain Streptococcus species are also reportedly responsiblefor cases of meningitis, bacterial pneumonia, endocarditis, erysipelas,and necrotizing fasciitis (also known as flesh-eating bacterialinfections). In the medical setting, the most important groups are the

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating blood borneinfectious diseases such as bacteremia, which may be caused by a numberof pathogenic organisms.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein are useful for treating diseases such asrespiratory tract infects (RTIs), including community acquired RTIs,such as community acquired pneumonia (CAP), community acquired bacterialpneumonia (CABP), and nosocomial RTIs, sinusitis, chronic bronchitis,pharyngitis, otitis media, asthma, and the like, skin and skin structureinfections (SSSIs), including both complicated and uncomplicated SSSI,and the like, urinary tract infections (UTIs), including uthreitis,gonorrhea, non-GC urethritis, prostatitis, and the like, blood-bornebacterial infections, such as bacteremia, and the like, traveler'sdiarrhea, and others.

In each of the foregoing embodiments, it is to be understood that thecompounds, compositions, methods, and medicaments described herein, maybe used in treating diseases arising from various organisms that areresistant to other compounds, including organisms that are resistant topenicillins, cephalosporins, quinolones, vancomycin, and/or macrolides,including ketolides, such as TEL and CTH. Illustratively, describedherein are compounds, compositions, methods, and medicaments useful fortreating diseases caused by clinically important mycoplasmal andureaplasmal species of humans, including AZI and TEL-resistant M.pneumoniae, doxycycline-resistant M. hominis, and doxycycline-resistantUreaplasma spp.

In each of the foregoing embodiments, the methods include the step ofadministering one or more compounds of formula (I), and/or any of thevarious subgenera of formula (I) described herein, and/or apharmaceutically composition of any one or more of the foregoingcompounds. The compound(s) and/or composition(s) are administered to apatient suffering from or in need of relief from a disease caused atleast in part by one or more of the organisms described herein. Inanother embodiment, the disease is caused at least in part by one ormore of the organisms exhibiting resistance to one or more macrolideantibiotics. In another embodiment, the disease is caused at least inpart by one or more of the organisms exhibiting resistance to one ormore ketolide antibiotics. In another embodiment, the disease is causedat least in part by one or more organisms selected from MRSA, MRSA 300(CA), VRSA, Macrolide-Lincosamide-Streptogramin B (MLS_(B)) resistantorganism, MDR pneumococcus, S. pneumoniae serotype 19A (also known asMultidrug-Resistant Pneumococcal Serogroup 19A), erythromycin resistantS. pyogenes, erythromycin resistant staphylococci, or S. pneumoniae withat least one erm(B), erm(A), mef(A), mef(E), mef(I), erm(B)+mef(A), L4,or 23S ribosomal protein mutation, or a combination thereof. In anotherembodiment, the disease is caused at least in part by one or more S.pyogenes. In another embodiment, the disease is caused at least in partby one or more S. pneumoniae. In another embodiment, the disease iscaused at least in part by one or more organisms selected from S.pyogenes and a S. pneumoniae with any one or more of erm(B), erm(A),mef(A), mef(E), mef(I), erm(B)+mef(A), L4, or 23S ribosomal proteinmutation, and combinations thereof. In another embodiment, the diseaseis caused at least in part by one or more organisms selected from anerythromycin resistant S. pyogenes, and a S. pneumoniae with at leastone erm(B), erm(A), mef(A), mef(E), mef(I), erm(B)+mef(A), L4, or 23Sribosomal protein mutation, and combinations thereof. In anotherembodiment, the disease is caused at least in part by one or moreorganisms selected from a S. pneumoniae with at least one erm(B),erm(A), mef(A), erm(B)+mef(A), L4, or 23S ribosomal protein mutation,and combinations thereof. In another embodiment, the disease is causedat least in part by one or more organisms resistant to CLR. In anotherembodiment, the disease is caused at least in part by one or moreorganisms resistant to AZI. In another embodiment, the disease is causedat least in part by one or more organisms resistant to TEL. In anotherembodiment, the disease is caused at least in part by one or moreorganisms resistant to CTH.

In each of the foregoing embodiments, the methods include the step ofadministering one or more compounds of formula (I), and/or any of thevarious subgenera of formula (I) described herein, and/or apharmaceutically composition of any one or more of the foregoingcompounds to a patient suffering from or in need of relief from adisease caused at least in part by one or more organisms selected fromGonococcus, such as Neisseria gonorrhoeae Mycoplasma, such as M.pneumoniae, M. genitalium, M. fermentans, and M. hominis, Ureaplasma,including U. parvum and U. urealyticum, Moraxella, including M.catarrhalis, Enterococcus, including E. faecalis, Staphylococcus,including S. aureus, S. epidermidis, MSSA, MRSA, MRSA 300, and VRSA,Streptococcus, including S. pneumoniae, such as serotype 19A S.pneumoniae, erythromycin resistant S. pyogenes, coagulase-negativestaphylococci, Emerging TEL-Resistant β-Haemolytic Streptococci, H.haemolyticus, including beta haemolytic streptococci, S. mitis, andviridans group strep, Chlamydia, including C. pneumoniae and C.trachomatis, H. influenzae, H. parainfluenzae, Legionella, such as L.pneumophila, Listeria, such as L. monocytogenes, Neisseria, such as N.meningitides, Mycobacterium leprae, Bacillus spp., Corynebacterium spp.,Micrococcus spp., and Anaerobic organisms.

It has been unexpectedly discovered that compounds like CEM-101 showsimilar activity compared to TEL towards TEL susceptible isolates, butlower MICs towards TEL intermediate and TEL resistant isolates, such asin the Belgian collection of S. pneumoniae described herein withconfirmed CAP resistant to macrolides and ketolides, such as TEL. Thecompounds described herein exhibit the widest spectrum and best activityagainst RTI pathogens among all tested MLSB-ketolide agents, includingAZI. CLR, ERY, TEL, clindamycin (CLN), and Synercid® (SYN), andcomparable spectrum and activity to levofloxacin (LEV). For example,CEM-101 MIC values were at ≦0.5 and ≦4 μg/ml for S. pneumoniae, L.pneumophila, and H. influenzae, respectively.

Without being bound by theory, it is believed that the efficacyobservable with the compounds described herein may be due at least inpart to one or more features of the compounds, including theintracellular accumulation, intracellular activity, distribution to manycompartments at high concentration, high activity on resistantorganisms, low potential for and low inducible resistance, andbactericidal activity of the compounds described herein. It wassurprisingly found herein that the compounds described herein are notPGP substrates or are poor PGP substrates.

The ketolides differ structurally from the macrolides, (1) lacking the3-O-cladinose, (2) including the presence of 3-keto group, and (3) inthe case of the compounds described herein, including the presence of anaromatic functionality to interact with domain II of the bacterialribosome. It is believed herein that in addition to the otherinteractions common to macrolides, ketolides, such as the compoundsdescribed herein, have additional interactions with the ribosome,including H-bonding to the 2-fluoro when present, interactions with the3-oxo group, and interactions with the pendant aromatic group attachedto the C-11, C-12 cyclic carbamate. Without being bound by theory, it isbelieved that these features may contribute to activity againstresistance orgs, the low observed inducible resistance, and/or thebactericidal activity of the compounds described herein.

In another embodiment, compounds are described herein that are activeagainst macrolide, including ketolide, resistant organisms, and alsohave a low potential for resistance development, and/or low inducibleresistance rates. Without being bound by theory, it is believed hereinthat the compounds bind more tightly to the ribosome of the infectingorganism. In addition, though again not being bound by theory, it isbelieved herein that the compounds bind to the ribosome of the infectingorganism more tightly and/or in a manner different from other macrolidesor even other ketolides. In particular, it is believed herein that thecompounds described herein bind to the ribosome with a differentorientation. In addition, it is believed herein that the compoundsdescribed herein have an additional binding site to the ribosome thatother macrolides or ketolides, allowing for tighter binding. In each ofthe foregoing, it is believed herein that the 1,2,3-triazole isresponsible for the differences in binding interactions between thecompounds described herein and other macrolides and ketolides.

For example, a common mechanism that leads to macrolide resistanceinvolves Erm-mediated methylation of a key bacterial ribosomalnucleotide (A2058EC) found in domain V of the 23S RNA. This nucleotidewas shown to play a major role in the ribosomal binding of macrolideantibiotics through interactions with the macrolide desosamine sugar.Methylation of A2058 effectively interferes with the binding of thedesosamine sugar thereby reducing the binding affinity of the macrolideto the bacterial ribosome. A possible explanation for the enhancedactivity of the compounds described herein against macrolide-resistantbacteria stems from additional domain II interactions through thependant aryl-1,2,3-triazolyl side chain. It has been suggested that thisadditional interaction helps compensate for alterations at the domain Vbinding site in resistant bacteria. In addition, though without beingbound by theory, and unlike other macrolides or ketolides, it isbelieved that the compounds described herein are able to bind bothdomain II and V of ribosomal RNA, and able to maintain in vitro activityagainst macrolide-resistant M. pneumoniae that have altered bindingsites in domain V due to the A2063G mutation. For example, one suchcompound, CEM-101 has lower MICs than TEL for the human mycoplasmas.CEM-101 maintained in vitro activity against two AZI resistant M.pneumoniae with MICs of 0.5 μg/ml while TEL MICs of 4 μg/mL exceeded thebreakpoint of 1 μg/mL used to designate susceptibility for otherbacterial species. The difference between the MIC₅₀ for the M.pneumoniae group overall and the MICs for the two resistant isolates wasthe same at 14 two-fold dilutions for each drug. Therefore, the modestlowering of the CEM-101 in vitro MIC may be more profound in vivo whentreating diseases caused at least in part by mycoplasmas. It was alsosurprisingly found that both CEM-101 and CEM-103 (3-cladinose of 101)show considerable binding to the ribosome erm-dimethylated at A2058.CEM-103 is the macrolide analog of CEM-101 having a 3-cladinose ratherthan a 3-oxo group, and therefore supports the theory that the1,2,3-triazole is responsible for the differences in bindinginteractions between the compounds described herein and other macrolidesand ketolides.

In addition, the compounds described herein have low potential forand/or low inducible resistance. In single step mutational resistanceand also in multi-step passage to resistance, no growth of mutants wasobserved when the strains were exposed to 4×, 8× and 16×CEM-101 MIC withany of the 4 strains tested. The mutation rates by organism were: E.faecium at <4.0×10⁻⁹ , S. aureus at <6.0×10⁻⁹ and S. pneumoniae at<6.4×10⁻⁸ to <1.4×10⁻⁹. Among 18 isolates tested for resistanceselection during passaging in sub-inhibitory concentrations ofantimicrobial agents, no significant variation (more than ±one log₂dilution) of the MIC values of CEM-101 was observed with eight strains(44.4%), including one S. aureus, three enterococci, three S.pneumoniae, and one βhemolytic Streptococcus. The remaining 10 strainsexhibited modest increases of CEM-101 MICs of four-fold (seven strains)to eight-fold (three strains), with no reversion or only a two-folddecrease in the MIC value after three consecutive daily passages inantimicrobial free media. Resistance selection during passaging insub-inhibitory concentrations was less overall for CEM-101 when comparedto other agents evaluated.

In another embodiment, compounds are described herein that are activeintracellularly. It has also been discovered herein that theintracellular accumulation and intracellular activity oftriazole-containing macrolides was not affected by Pgp or MultidrugResistant Protein (MRP) inhibitors. Accordingly, it is believed that thecompounds described herein are not substrates or are poor substrates ofP-glycoprotein (plasma or permeability glycoprotein, Pgp). It isappreciated that Pgp is an efflux mechanism that may lead to resistanceby some organisms against certain antibiotics, such as has been reportedfor AZI and ERY in macrophages in which both antibiotics are substratesof the P-glycoprotein. Accordingly, it has been surprisingly found thatthe compounds described herein accumulate intracellulary. In addition tothe intracellular accumulation, it has been surprisingly discovered thatthe triazole-containing macrolide and ketolide compounds describedherein have high intracellular activity. It has also been surprisingfound herein that the compounds described herein have lower proteinbinding than is typical for macrolides at lower pH, such as the pH foundin bacterial infections, including but not limited to abscesses. It isappreciated that the lack of intracellular activity typically observedwith anti-bacterial agents, including other macrolides and ketolides,may be due to high protein binding, and/or to the relatively lower pH ofthe intracellular compartments, such as is present in abscesses.

However, even when not removed by active efflux, the concentration ofother anti-bacterial agents, including other macrolides and ketolides,in macrophages may not be efficacious in treating disease because of thelow pH of the lysozomal compartment. For example, the acidic environmentprevailing in the phagolysosomes (where S. aureus sojourns during itsintracellular stage) may impair the activity of antibiotics, such as theAZI, CLR and TEL. It has been unexpectedly found that the compoundsdescribed herein retain their anti-bacterial activity at low pH. It isappreciated that the intracellular activity of the compounds describedherein may be an important determinant for fast and complete eradicationand, probably also, for prevention of resistance in the target organism.

Lack of effective antimicrobial therapy results in intracellularsurvival of bacteria, which remains a major cause of bacterialspreading, life-threatening therapeutic failures, and establishment ofchronic, relapsing infections. These situations are observed during thecourse of infections caused by many organism, including meningitis fromL. monocytogenes, invasion of lung macrophages from L. pneumophila, andendocarditis, osteomyelitis, and skin and skin structure infections fromS. aureus.

While it has been reported that intracellular accumulation of anantibiotic is indicative of efficient activity against bacteria,pharmacodynamic evaluation of a large series of commonly usedantibiotics has revealed that other parameters such as intracellularbioavailability and modulation of activity in the infected compartmentare also important. The observations described herein confirm and extendprevious observations made with macrolides in this context due to thesurprising differential behavior exhibited by the triazole-containingmacrolides described herein, compared to known macrolide and ketolides,such as TEL, AZI, and CLR.

It is surprisingly found that triazole-containing macrolides accumulateto a considerably larger extent than the comparators, including AZI, andconsistently expresses greater potency (decreased values of E₅₀ andC_(s)) while showing similar maximal efficacy (E_(max)) to comparators.Without being bound by theory, it is believed that this indicates thatthe improvements resulting from the structural modifications introducedin CEM-101 relate to modulation of pharmacokinetic properties andintrinsic activity (including its reduced susceptibility tophysico-chemical conditions prevailing in the infected compartment)rather than to a change in its mode of action. Thus, triazole-containingmacrolides exhibit the essentially bacteriostatic character ofmacrolides, but express it better in the intracellular milieu and atconsiderably lower extracellular concentrations than the comparators.

Without being bound by theory, it is believed that the cellularaccumulation of triazole-containing macrolides, such as CEM-101, resultsfrom the general mechanism of proton trapping of weak organic basesenvisaged for all macrolides as accumulation is almost completelysuppressed, in parallel with AZI, by exposure to acid pH or to theproton ionophore monensin. Based on the general model ofdiffusion/segregation of weak bases in acidic membrane-boundcompartments, accumulation is determined by the number of ionizablegroups and the ratios between the membrane permeability coefficients ofthe unionized and ionized forms of the drug. While CEM-101 has twoionizable functions, the pKa of the aminophenyltriazole is calculated tobe less than 4, suggesting that the molecule is largely monocationic(similar to CLR and TEL) at neutral and even at lysosomal pH 5). Incontrast, AZI has two ionizable functions with pK_(a)s>6 and istherefore dicationic intracellularly. CEM-101, however, possesses afluoro substituent in position 2, which should make it more lipophilicthan CLR or TEL. Without being bound by theory, it is believed that theratio of the permeability constants of the unionized and ionized formsof CEM-101 in comparison with LR or TEL may be as important as thenumber of ionizable functions to determine the level of cellularaccumulation of weak organic bases. Without being bound by theory, it isbelieved that the greater cellular accumulation of CEM-101 may bepartially due to its lack of susceptibility to Pgp-mediated efflux(which is expressed by THP-1 macrophages under our culture conditions)in contrast to AZI.

It has been observed that many known macrolides have a large volume ofdistribution, which it is believed is related to their ability toaccumulate inside eukaryotic cells by diffusion/segregation in acidiccompartments, namely lysosomes and related vacuoles. As a consequence,known macrolides had been considered candidates for the treatment ofinfections localized in these compartments. Thus, it might be assumedthat macrolides are suitable for treating infections caused by typicalintracellular pathogens such Legionella and Chlamydia, based on a largearray of both in vitro and clinical data. However, direct quantitativecomparisons between intracellular and extracellular activities usingfacultative intracellular pathogens, such as S. aureus or L.monocytogenes, suggest that known macrolides express only a minimalfraction of their antibacterial potential intracellularly, especiallyconsidering their great intracellular accumulation. This minimizedantibacterial potential against organisms replicating in phagolysosomesand related vacuoles is believed to be related to acidic pH which isknown to reduce the activity of known macrolides. Another factor is thatsome organisms, such as L. monocytogenes, may actually replicate inother subcellular compartments. In addition, certain macrolides, such asAZI, are subject to active efflux from macrophages, which furthercontributes to suboptimal intracellular activity.

In contrast, the cellular accumulation and intracellular activity of thetriazole-containing compounds described herein, using models that havebeen developed for the study of the intracellular pharmacodynamics ofantibiotics, is substantially improved over known macrolides, includingketolides. Thus, the compounds described herein maintain the maximalefficacy of their MICs, and show greater potency against intracellularforms of for example, Staphylococcus, Listeria, and Legionella comparedto TEL, AZI, and CLR. Without being bound by theory, it is believed thatthis improved intracellular potency of the triazole-containing compoundsdescribed herein results from the combination of the higher intrinsicactivity against Staphylococcus, Listeria, and Legionella coupled withthe retained activity at low pH, and the ability to distribute to a widevariety of intracellular compartments.

In another embodiment, the triazole-containing macrolide and ketolidecompounds have intracellular activity, such as intracellular activityagainst Staphylococcus, such as S. aureus. Survival of S. aureus withineukaryotic cells is critical for the persistence of infection. It isappreciated that routine susceptibility testing are usually determinedagainst extracellular bacterial only, and therefore may be misleading intheir prediction of efficacy against intracellular organisms. In anotherembodiment, compounds, compositions, methods, and uses are describedherein for treating a disease caused at least in part by anintracellular Staphylococcus infection. In another embodiment, thedisease caused by the Staphylococcus infection is community acquiredMRSA (CA-MRSA), community acquired pneumonia (CA-P), or a skin and skinstructure infection (SSSIs). It is further appreciated that S. aureus isconsidered a virulent strain, and thus treatment with bacteriostaticagents may be ineffective. For example, recurrence may be a problem whentreating such strains. It has been unexpectedly discovered herein thatthe compounds described herein are also bactericidal and thereforeuseful in treating diseases caused by Staphylococcus, and in particularintracellular Staphylococcus, such as S. aureus in either instance.

In another embodiment, the triazole-containing macrolide and ketolidecompounds have intracellular activity, such as intracellular activityagainst Listeria, such as L. monocytogenes. In another embodiment, thetriazole-containing macrolide and ketolide compounds have intracellularactivity, such as intracellular activity against Legionella, such as L.pneumophila.

In another embodiment, the triazole-containing macrolide and ketolidecompounds have intracellular activity against Mycobacterium, such as M.leprae. In another embodiment, compositions, methods, and medicamentsare described herein for treating diseases caused at least in part by M.leprae, including but not limited to Hansen's disease (leprosy). In oneaspect, the compositions, methods, and medicaments include atherapeutically effective amount of one or more compounds describedherein. In another embodiment, compounds, compositions, methods, andmedicaments are described herein for treating diseases caused at leastin part by M. leprae that are resistant to CLR. For example, CEM-101 hasbeen found to be 2-4 times more active than other antibiotics of thesame class mainly CLR and TEL. It is active against a variety ofmacrolide resistant pathogenic strains of S. aureus, S. pyogenes, and S.pneumoniae.

In another embodiment, the compounds, methods, and medicaments describedherein include a therapeutically effective amount of one or morecompounds described herein, wherein the therapeutically effective amountis an amount effective to exhibit intracellular antibacterial activity.

In another embodiment, compounds are described herein that arebactericidal. In another embodiment, the compounds, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidalactivity, including in vivo bactericidal activity. It has been reportedthat macrolides are generally bacteriostatic. Bacteriostatic compoundsdo not kill the bacteria, but instead for example inhibit growth andreproduction of bacteria without killing them; killing is accomplishedby bactericidal agents. It is understood that bacteriostatic agents mustwork with the immune system to remove the microorganisms from the body.Bacteriostatic antibiotics may limit the growth of bacteria via a numberof mechanisms, such as by interfering with bacterial protein production,DNA replication, or other aspects of bacterial cellular metabolism. Incontrast, bactericidal antibiotics kill bacteria; bacteriostaticantibiotics only slow their growth or reproduction. Penicillin is abactericide, as are cephalosporins, all belonging to the group ofβ-lactam antibiotics. They act in a bactericidal manner by disruptingcell wall precursor leading to lysis. In addition, aminoglycosidicantibiotics are usually considered bactericidal, although they may bebacteriostatic with some organisms. They act by binding irreversibly to30s ribosomal subunit, reducing translation fidelity leading toinaccurate protein synthesis. In addition, they inhibit proteinsynthesis due to premature separation of the complex between mRNA andribosomal proteins. The final result is bacterial cell death. Otherbactericidal antibiotics include the fluoroquinolones, nitrofurans,vancomycin, monobactams, co-trimoxazole, and metronidazole.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidal activityagainst one or more pneumococcus. It has been reported that resistanceby pneumococcus occurs very rapidly. Accordingly, without being bound bytheory, it is believed herein that treating such diseases usingbacteriostatic agents may be unsuccessful in two respects. First, simplystopping the progression of the disease with a bacteriostatic agent maybe insufficient because the immune system may not intervene to assist incuring the disease at a necessary level. For example, some bacterialorganisms are not killed by the immune system because they reside inintracellular compartments. Thus, once the treatment course has ended,rapid recurrence of disease may result. Second, because some portion ofthe bacterial population will likely be eliminated, the remainingpopulation may be selected for resistance development. It is believedherein that an intracellularly active agent, and/or an intracellularlyactive and bactericidal agent, will be efficacious in treating suchdiseases. In one illustrative embodiment, compounds described hereinthat achieve an intracellular concentration of 20× the MIC of thetargeted bacteria. It has been reported that most, if not all, macrolideantibiotics, though bactericidal in vitro, are only bacteriostatic invivo. For example, as described hereinbelow, when the time between thelast dose of compound was extended, the bioload reduction levelsremained the same for the triazole-containing compounds describedherein, indicating a bactericidal response. In contrast, the TEL and CLRdose groups demonstrated bioload increases when the time interval wasextended. Thus, those latter two macrolide/ketolide agents demonstrateda more classical bacteriostatic response.

In another embodiment, compounds, compositions, methods, and medicamentsare described herein having a long post-antibiotic effect (PAE). Inanother embodiment, compounds, compositions, methods, and medicamentsare described herein that are synergistic with other anti-bacterialagents. In another embodiment, the other anti-bacterial agents areselected from aminoglycoside antibiotics, cephalosporins, anddihydrofolate reductase and dihydropteroate synthetase inhibitors, suchas gentamicin (GEN), ceftriaxone (CRO), trimethoprim/sulfamethoxazole(TMP/SMX).

In each of the forgoing embodiments, it is to be understood that thedisease treatable by the compounds, compositions, and methods describedherein is caused by at least one organism other than one or more of thefollowing: S. aureus (ATCC 29213, MSSA, MLS-S), E. faecum (ATCC 19434),K. pneumonia (13883), E. coli (ATCC 25922), S. typhimurium (ATCC 14028),S. pneumonia (ATCC 49619), S. pyogenes (ATCC 19615), S. pneumonia (163,Mef A), S. pneumonia (303, ErmB), S. aureus (MRSA, 33591), H. influenzae(ATCC 49247), S. pneumonia (3773, ErmB), S. pneumonia (5032), S.pyogenes (1721), S. pyogenes (1850), S. pyogenes (3029), and S. pyogenes(3262).

In another illustrative embodiment, compounds of Formula (I) aredescribed herein where X and Y are taken together with the attachedcarbon to form a C(O) group. In another embodiment, X is H, Y is OR⁷,where R⁷ is a monosaccharide radical, such as cladinosyl. In anotherembodiment, compounds of Formula (I) are described herein where W isfluoro. In another embodiment, compounds of Formula (I) are describedherein where A and B are taken together to form an alkylene group,including but not limited to propylene, butylene, and pentylene. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form butylene. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene. In another embodiment, compounds of Formula(I) are described herein where A and B are taken together to formbutylenes and C is 2-pyridinyl or aminophenyl, such as 3-aminophenyl. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form propylenes, butylenes, or pentylenes;and C is aminophenyl, such as 3-aminophenyl. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene and C is 3-pyridinyl or benzotriazole. Inanother embodiment, compounds of Formula (I) are described herein whereC is an optionally substituted aryl or heteroaryl group. In anotherembodiment, compounds of Formula (I) are described herein where V is acarbonyl group. In another embodiment, compounds of Formula (I) aredescribed herein where R¹⁰ is hydrogen. In another embodiment, X is H, Yis OR⁷, where R⁷ is a monosaccharide radical, such as cladinosyl, and Cis 3-pyridinyl or benzotriazolyl.

In another embodiment, C is optionally substituted phenyl, such asphenyl, halophenyl, haloalkylphenyl, aminophenyl, and the like,optionally substituted pyridinyl, such as 2-pyridinyl and 3-pyridinyl,optionally substituted benzotriazole, and the like.

In another embodiment, A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group.

In another embodiment, compounds described in any of the precedingembodiments wherein V is C(O) are described. In another embodiment,compounds described in any of the preceding embodiments wherein W is Hor F are described. In another embodiment, compounds described in any ofthe preceding embodiments wherein A is CH₂, B is (CH₂)_(n), and n is aninteger from 2-4 are described. In another embodiment, compoundsdescribed in any of the preceding embodiments wherein C is aryl orheteroaryl are described. In another embodiment, compounds described inany of the preceding embodiments wherein C is 3-aminophenyl or3-pyridinyl are described. In another embodiment, compounds described inany of the preceding embodiments wherein R₁₀ is hydrogen. In anotherembodiment, compounds described in any of the preceding embodimentswherein A and B are taken together to form butylene or pentylene, and Xand Y are taken together with the attached carbon to form a C(O) group.In another embodiment, compounds described in any of the precedingembodiments wherein A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group, and W is F.

In another embodiment, an antibacterial composition is described herein,wherein the composition includes an effective amount of one or morecompounds described herein, and a pharmaceutically acceptable carrier,excipient, or diluent therefor, or a combination thereof.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. Illustratively,compositions may include one or more carriers, diluents, and/orexcipients. The compounds described herein may be formulated in atherapeutically effective amount in conventional dosage forms for themethods described herein, including one or more carriers, diluents,and/or excipients therefor. Such formulation compositions may beadministered by a wide variety of conventional routes for the methodsdescribed herein in a wide variety of dosage formats, utilizingart-recognized products. See generally, Remington's PharmaceuticalSciences, (16th ed. 1980). It is to be understood that the compositionsdescribed herein may be prepared from isolated compounds describedherein or from salts, solutions, hydrates, solvates, and other forms ofthe compounds described herein. It is also to be understood that thecompositions may be prepared from various amorphous, non-amorphous,partially crystalline, crystalline, and/or other morphological forms ofthe compounds described herein.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known in the medical arts.

In one embodiment, the compounds described herein are administered to ahuman orally at a dose of about 1 to about 10 mg/kg, about 2 to about 8mg/kg, or about 4 to about 6 mg/kg of patient body weight. In anotherembodiment, the daily adult human dose is about 100 to about 1,000 mg,which may be administered qd, bid, tid, and the like. In anotherembodiment, the daily adult human dose is about 400 to about 600 mg,which may be administered qd, bid, tid, and the like. Such doses may beadministered, once, twice, or thrice per day. Illustrative oral unitdosages are 50, 100, 200, and 400 mg (single or divided). Without beingbound by theory, it is believed that such illustrative dosages aresufficient to achieve plasma levels of about 1 μg/mL, which may besufficient to observe bactericidal activity of the compounds describedherein, such as for macrolide-susceptible pneumococci. It is appreciatedthat as described herein, the compounds described herein, includingCEM-101, reach high concentration in tissues, such as lung tissues.Without being bound by theory, it is believed herein that the compoundsdescribed herein, including CEM-101, may achieve tissue levels that areat least about 10-times the MIC for strains, includingmacrolide-resistant strains, such as but not limited to H. influenzaeand H. influenzae resistant to AZI, S. pyogenes and S. pneumoniaeresistant to any one of AZI, CLR, CTH, and/or TEL, and other resistantorganisms described herein.

The compounds described herein may be prepared as described herein, oraccording to US Patent Application Publication No. 2006/0100164 and inPCT International Publication No. WO 2009/055557, the disclosures ofwhich are incorporated herein by reference in their entirety.

Briefly, the synthesis of triazole containing ketolides begins with theknown two step preparation of the 12-acyl-imidazole intermediate 4(Scheme I) from CLR (2). Intermediate 4 is converted into the11,12-cyclic carbamates 5a-c by the reaction with the corresponding 3-,4- or 5-carbon linked amino alcohols. Treatment of 5a-c with tosylchloride provides tosylates 6a-c. Displacement of the tosyl group withNaN₃ gives the corresponding azido compounds 7a-c. Cleavage of thecladinose sugar of 7a-c to 8a-8c is accomplished by treatment with HClin MeOH. Swern oxidation of the 3-hydroxy group of 8a-c gives thecorresponding protected ketolides 9a-c which are subsequentlydeprotected with methanol to afford the required azido ketolides 10a-c,respectively. These azido compounds were reacted withterminally-substituted alkynes in the presence of copper iodide intoluene at 60° C. to regio-selectively afford the corresponding4-substituted-[1,2,3]-triazoles 11a-18a, 11b-18b, and 11c-18c.

The azide of intermediates 10a-c is converted to the4-substituted-[1,2,3]-triazoles via a cycloaddition reaction withsubstituted acetylenes. Triazole rings may be formed via a Huisgen 1+3cycloaddition reaction between an azide and an alkyne resulting in amixture of 1,4- and 1,5-regioisomers as depicted in Route A of SchemeII. Alternatively, the procedure of Rostovtsev et al.⁸ may be followedusing the addition of a CuI catalyst to the reaction to selectively orexclusively produce the 1,4-regioisomer as depicted in Route B of SchemeII.

The triazole ring side chain is also incorporated into the CLR ringsystem. In one embodiment, a butyl alkyl side chain is chosen. It isappreciated that many butyl side chain analogs in the ketolide serieshave improved antibacterial activity based on in vitro MIC results.Intermediate 7b is directly converted into the4-substituted-[1,2,3]-triazole via copper catalyzed cyclization withterminally substituted acetylyenes, as shown in Scheme III. The acetateprotecting groups of 19a-e are removed with LiOH in methanol to affordthe corresponding 4-substituted-[1,2,3]-triazoles 20a-e.

Substitution of the 2-position hydrogen with a fluorine is accomplishedby electrophilic fluorination of 9b (Scheme IV) using Selectfluor®. Theazido group of intermediate 22 is converted to a series of4-substituted-[1,2,3]-triazoles 23a-b via the standard conditions.

In another embodiment, the following compounds are described:

Minimum inhibitory concentration (μg/mL)^(a) S. aureus 96:11480 S.pneumoniae H. influenzae 29213 Ery-R 49619 163 303 49247 Entry R n Ery-S(MLSb) Ery-S Ery-R (MefA) Ery-R (ermB) Ery-S TEL  ≦0.125  ≦0.125 ≦0.125 ≦0.125  ≦0.125    4 AZI  ≦0.125 >64 ≦0.125 >64 >64    2 11a 11b 11c

3 4 5    1  ≦0.125  ≦0.125    1  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125 >64    2    0.25 >64    8   16 12a 12b 12c

3 4 5   0.25 ≦0.125 ≦0.125   0.5  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125    8    8    0   64    8   16 13a 13b 13c

3 4 5   1   0.25   0.5    2    0.25    1 ≦0.125 ≦0.125 ≦0.125  ≦0.125 ≦0.125    0.5   16    8    2 >64    8   64 14a 14b 14c

3 4 5   2 ≦0.125 ≦0.125    2  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125    0.5 ≦0.125  ≦0.125 >64  ≦0.125    0.25 >64    4   64 15a 15b 15c

3 4 5   2 ≦0.125 ≦0.125    2    4    0.25 ≦0.125 ≦0.125 ≦0.125    1    2   0.25 >64   64    4 >64   64   16 16a 16b 16c

3 4 5   0.5 ≦0.125 ≦0.125 nt  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125 >64  ≦0.125    0.25   16    2    8 17a 17b 17c

3 4 5   1 ≦0.125   0.25    1  ≦0.125    0.5 ≦0.125   0.12 ≦0.125  ≦0.125   0.12  ≦0.125 >64    1    2 >64   16   32 18a 18b 18c

3 4 5   1   1 ≦0.125    2    2  ≦0.125 ≦0.125 ≦0.125 ≦0.125    0.5    4 ≦0.125 >64   64   64 >64   32    8 ^(a)National Committee for ClinicalLaboratory Standards. Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria that Grow Aerobically, 6^(th) ed.; Approved standard:NCCLS Document M7-A6, 2003.

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae H. influenzae 25923 49619 163 303 49247 Entry REry-S RN220 Ery-S Ery-R (MefA) Ery-R (ermB) Ery-S TEL ≦0.25 2 ≦0.125≦0.125 ≦0.125 4 20a

0.25 8 ≦0.0625 0.125 2 NT 20b

0.25 8 ≦0.0625 ≦0.06 1 NT 20c

1 8 ≦0.0625 0.5 2 NT 20d

1 8 ≦0.0625 0.5 2 NT 20e

≦0.25 8 ≦0.0625 0.5 2 NT

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae 96:11480 Ery 163 303 H. influenzae 29213 R 49619Ery -R Ery-R 49247 Entry R Ery-S (MLSb) Ery-S (MefA) (ermB) Ery-S TEL≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 4 AZI ND ≦0.125 >64 ≦0.125 >64 >6423a

≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2 23b (CEM- 101)

≦0.06 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2

In each of the foregoing embodiments, the primary screening panelconsisted of relevant Staph. aureus, S. pyogenes, S. pneumoniae(including strains resistant to AZI and TEL). MICs against all pathogenswere determined using broth microdilution method as per NCCLSguidelines. Compounds described herein, such as CEM-101 were found to behighly potent having MICs against S. pneumoniae (3773) of ≦0.125 μg/mLand S. pyogenes (1850) of 0.5 μg/mL, compared to 1 and 8 μg/mL,respectively for TEL. CEM-103 (20c), an analogue of CEM-101 thatcontains the 3-O-cladinose was found to be less active.Non-heteroaromatic substituted triazole containing ketolides were lessactive.

The ketolides were tested against erythromycin-sensitive (Ery-S) anderythromycin-resistant (Ery-R) strains of S. aureus (29213 (Ery-S) and96:11480 (Ery-R)), S. pneumoniae (49619 (Ery-S) and 163 and 303 (Ery-R))and H. influenzae (49247 (Ery-S)) (Tables 1-3). The broth micro-dilutionmethod was used to determine the Minimum Inhibitory Concentrations(MICs) against all pathogens as per the Clinical and LaboratoryStandards Institute (CLSI).

The chain length of the alkyl side chain affected activity (Table 1).For example, the 3-carbon linked phenyl substituted triazole 11a wasless active against Ery-S and Ery-R S. aureus and was inactive againstEry-R S. pneumoniae 303 (ermB) a the tested concentrations, whereas thecorresponding 4- and the 5-carbon linked phenyl substituted triazoles11b and 11c were more active against these organisms. A similar trendwas observed for the 2-pyridyl substituted triazoles 14a-c, the3-amino-phenyl substituted triazoles 16a-c, and the 2,5-dichlorophenoxysubstituted triazoles 17a-c.

The 4-carbon linked 2-pyridyl substituted triazole 14b and the3-amino-phenyl substituted triazole 16b possessed the highest potencyagainst S. pneumoniae 303, both having MIC values (≦0.125 μg/mL)comparable to TEL. The ketolide containing the 4-carbon linked 3-pyridylsubstituted triazole 15b was less active against this strain (MIC of 64μg/mL). Within this series antibacterial activity was improved byextending the carbon linker to 5 atoms, for example the MIC against S.pneumoniae 303 for compound 15c improved from 64 to 4 μg/mL. A similareffect was also observed for the benzo-triazole containing ketolide 18cagainst S. aureus but 18c was still inactive against S. pneumoniae 303.It is appreciated that a balance between the length of the linker andnature of the aromatic substitution of the triazole may affect theoverall activity against macrolide resistant S. pneumonia and S. aureus.

A correlation between linker length and activity was also observed forH. influenzae (49247) where the most potent ketolide series had thesubstituted triazole linked through either a 4-carbon (11b-14b, 16b,17b) or a 5-carbon (15c, 18c) chain. Interestingly, the most potentaromatic series against H. influenzae was the 3-amino-phenyl with a 3-,4- or 5-carbon linker (16a, 16b, 16c) having MICs of 16, 2, and 8 μg/mL,respectively,

The macrolides containing a cladinose at the 3 position were all highlyactive against Ery-S S. pneumoniae (49619) (Table 2). However, theseanalogs were less potent than TEL against Ery-R strains. The MICs weresignificantly higher for the cladinose containing analogs with either2-pyridyl, 2-aminophenyl or 2,6-dichlorophenyl triazole substituentsthan for the corresponding ketolides (20a, 20c, and 20d versus 14b, 16b,and 17b). Conversely, antibacterial activity was re-established forketolide analogs 15b (3-pyridyl) and 18b (benzo-triazole) by replacingthe keto with the cladinose group in analogs 20b (3-pyridyl) and 20e(benzo-triazole). The MICs improved from 64 μg/mL for 15b and 18b to 1and 2 μg/mL for 20b and 20e, respectively. A similar activity trend wasalso observed for Ery-R S. pneumoniae 163 (MefA).

Compound Examples

A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-(3-dimethylamine-4-deoxy-6-O-acetyl-glu-copyranosyl)-2-fluoro-3-oxo-erythronolideA, 11,12-carbamate (15 mg, 0.019 mmol), 6-Ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), CuI (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 14 mg of the desired compound. MS:C₄₄H₆₆FN₇O₁₂ calculated M⁺=903.5. Found: M+H⁺=904.5.

11-N-4-(3-aminophenyl)-[1,2,3]triazol-1-yl]-butyl}-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate (CEM-101). A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (17 mg, 0.023 mmol), 3-Ethynyl-phenylamine (5.4 mg,0.046 mmol), CuI (1 mg, 0.005 mmol), and toluene (0.2 mL) was heated to70° C. After 16 h, the mixture was concentrated and directly subjectedto silica gel chromatography (9:1, chloroform:methanol plus 1% ammoniumhydroxide) to give 17 mg of the desired compound, MS C₄₃H₆₅FN₆O₁₀calculated M⁺=844.47. Found: M+H⁺=845.5

11-N-{4-[4-(6-Amino-pyridin-2-yl)-[1,2,3]triazol-1-yl]-butyl}-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (15 mg, 0.02 mmol), 6-ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), CuI (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 14 mg of the desired compound OP1357. MS:C₄₂H₆₄FN₇O₁₀ calculated M⁺=845.5. Found: M+H⁺=846.5.

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-O-dasosaminyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-O-desosaminyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.0039 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), CuI (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₀ calculated M⁺=866.5. Found: M+H+867.5.

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-mycaminosyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-mycaminosyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.004 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), CuI (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₁ calculated M⁺=882.5. Found: M+H⁺=883.5.

Method Examples

Example.

The in vitro activities of CEM-101 is compared with those of AZI, CLR,TEL and doxycycline against 10 isolates of C. pneumoniae and 10 strainsof C. trachomatis in HEp-2 cells. The MIC at which 50% and 90% of theisolates of C. pneumoniae are inhibited by CEM-101 was 0.25 μg/ml(range: 0.25 to 1.0 μg/ml). The MIC at which 50% and 90% of the strainsof C. trachomatis were inhibited was 0.25 μg/ml (range: 0.125 to 0.5μg/ml). The MIC90s for both C. trachomatis and C. pneumoniae againstAZI, CLR, TEL, and doxycycline were 0.125, 0.06, 0.06, 0.06 μg/ml,respectively. The MICs of CEM-101 were consistent from isolate toisolate, varying by only one or two dilutions, which is especiallyimpressive given the wide geographical distribution of the isolatestested. These results indicate that CEM-101 and the compounds describedherein is an effective antibiotic for treating C. trachomatis andrespiratory tract infections caused by C. pneumoniae.

Example.

36 M. pneumoniae were collected from the respiratory tract of adults andchildren with pneumonia between 1992 and 2006. 2 isolates collected fromchildren in Birmingham, Ala. in 2005 were macrolide-resistant (AZIMICs>32 μg/ml); both of which had been shown to have an A2063G mutationin domain V of the rRNA gene. 5 M. genitalium included reference strainsobtained from the urogenital tracts of patients in the United States (3isolates) and Denmark (2) isolates. 15 M. fermentans from therespiratory or urogenital tracts were obtained from the MycoplasmaCollection at the National Institutes of Health and patients inBirmingham, Ala. between 1992 and 2004. 13 M. hominis were obtained fromclinical specimens of the urogenital tract or wounds between 1994 and2007. 2 isolates were resistant to doxycycline (MICs 8-16 μg/ml). 10Ureaplasma parvum were obtained from urogenital specimens between 2002and 2005. 7 were doxycycline-resistant (MICs 4-16 μg/ml). 10 U.urealyticum were obtained from various urogenital tract specimens,placentas or neonatal respiratory secretions between 1990 and 2005. 4were resistant to doxycycline (MICs 4-32 μg/ml).

All mycoplasma and ureaplasma isolates were inhibited by CEM-101 atconcentrations<0.5 μg/ml, making it the most potent compound testedoverall. M. pneumoniae MICs for CEM-101 ranged from 0.000000063-0.5μg/ml with MIC90=0.000125, making its activity 4-fold greater than AZI,8-fold greater than TEL. LZD was inactive against M. pneumoniae, butsome M. fermentans and M. hominis had MICs<1 mg/ml. 2macrolide-resistant MP with AZI and TEL with MICs greater than 32 μg/mlwere inhibited by CEM-101 at 0.5 μg/ml. CEM-101 MBCs were greater than16-fold greater than MICs for 9 M. pneumoniae indicating the drug isbacteriostatic against this organism. CEM-101 was active against alldoxycycline-resistant M. hominis and Ureaplasma spp. Excluding 2macrolide-resistant M. pneumoniae, no isolate of any species tested hadan MIC greater than 0.063 μg/ml for CEM-101.

MIC Testing. Antimicrobial powders were dissolved as instructed by themanufacturer and frozen in 1 ml aliquots containing 2048 μg/ml. Drugstested included: CEM-101, AZI, TEL, doxycycline, levofloxacin, andlinezolid. A working dilution of each drug was prepared on the day ofeach assay based on the anticipated MIC ranges for each drug. Serial2-fold antimicrobial dilutions were performed in 10B broth forUreaplasma spp. and SP4 broth for Mycoplasma spp. in 96 well microtiterplates. For macrolides and ketolides tested against M. pneumoniae,dilutions were taken down to 0.000000063 μg/ml to measure the endpointMIC for these potent agents. 0.175 mL inoculum of 104-105 CCU/mlobtained by inoculation of organisms from frozen stock of knownconcentration into prewarmed broth and incubating 2 hrs at 37° C. wasadded to the drug dilutions. Plates were sealed, incubated aerobicallyat 37° C. in air and examined daily until a color change was detected inthe drug-free growth control. MIC=the lowest concentration of a drug inwhich the metabolism of the organisms was inhibited as evidenced by lackof color change at the time the drug free control first showed colorchange.

Quality Control. The inoculum of each isolate was verified by serialdilutions and plate counts. Quality control strains used to validateaccuracy of MICs for comparator antimicrobial agents included M.pneumoniae (UAB-834), M. hominis (UAB-5155) and U. urealyticum(UAB-4817), all of which are low passage clinical isolates for which a 3dilution MIC range has been established.

MIC Range MIC₅₀ MIC₉₀ μg/mL μg/mL μg/mL M. pneumoniae (36 isolates)CEM-101 ≦0.000000063-0.5        0.000032 0.000125 AZI ≦0.000016-≧32    0.00025 0.0005 TEL    0.000031-≧32    0.00025 0.001 Doxycycline0.016-0.25  0.125 0.25 Levofloxacin 0.125-1    0.5 0.5 Linezolid  32-12864 128 M. genitalium (5 isolates) CEM-101 ≦0.000032 NA NA AZI≦0.000032-0.005     NA NA TEL ≦0.00003-0.00025   NA NA Doxycycline≦0.008-0.031   NA NA Levofloxacin 0.125-1    NA NA Linezolid  4-128 NANA M. fermentans (15 isolates) CEM-101 ≦0.008 ≦0.008 ≦0.008 AZI0.125-1    0.5 0.5 TEL 0.002-0.031 ≦0.008 0.016 CLN ≦0.008-0.063   0.0160.031 Doxycycline 0.016-0.5  0.125 0.5 Levofloxacin ≦0.008-0.25    0.0310.125 Linezolid 0.5-4   1 4 M. hominis (13 isolates) CEM-101 0.002-0.0080.004 0.008 AZI 0.5-4   4 2 TEL 0.125-0.5  0.25 0.5 CLN ≦0.008-0.031  ≦0.008 0.016 Doxycycline ≦0.008-0.016   0.125 8 Levofloxacin 0.125-0.5 0.25 0.5 Linezolid 1-8 2 4 U. parvum (10 isolates) CEM-101 0.002-0.0310.008 0.016 AZI 0.5-4   2 4 TEL 0.008-0.063 0.063 0.125 Doxycycline0.031-16   8 16 Levofloxacin 0.125-2    0.5 2 Linezolid  128->256 >256 >256 U. urealyticum (10 isolates) CEM-101 0.004-0.0630.008 0.031 AZI 0.5-4   2 4 TEL 0.016-0.25  0.063 0.25 Doxycycline0.031-32   1 16 Levofloxacin 0.5-2   0.5 1 Linezolid   256->256 >256>256M. pneumoniae Macrolide MIC Distribution. All but two isolates testedagainst CEM-101 showed an MIC of 0.0001 μg/mL or less. In contrast, morethan half all isolates tested against AZI or TEL showed an MIC of 0.0001μg/mL or greater.

MBC Testing. 9 M. pneumoniae were tested to determine MBCs for CEM-101.Aliquots (30 μl) from each well that had not changed color at the timethe MIC was read were added to 2.97 mL broth (1:100 dilution) to makecertain drug is diluted below inhibitory concentration to allow livingorganisms to grow to detectable levels. Growth control was subculturedto ensure presence of viable organisms in the absence of drug. Brothswere incubated at 37° C. MBC=concentration of antimicrobial in which nogrowth was apparent by lack of color change in broth after prolongedincubation. MBCs for 9 M. pneumoniae were all >16-fold greater than thecorresponding MICs indicating CEM-101 is bacteriostatic against thisorganism.

Example.

An in vitro model of mycobacterial growth arrest was developed usingMycobacterium bovis BCG. When an exponentially growing culture wastransferred to an evacuated tube, growth continued; however, treatmentwith a source of nitric oxide (50 mM DETA-NO) halted growth immediately.Aeration restored growth. When the period of growth arrest exceeded 4hrs, a time lag occurred before aeration could restore growth. This lagtime was maximal after 16 hrs of growth arrest, at which point the lagbefore growth restoration was 24 hrs. Without being bound by theory, itis believed herein that these time lags may indicate that one transitionperiod was required to achieve full arrest of growth and another tofully recover from growth arrest. Without being bound by theory, it isalso believed herein that DETA-NO-induced growth arrest failed toprotect from the lethal effects of anaerobic shock, which may havecaused rapid cell lysis of both growing and growth-arrested cells. Whilegrowth arrest had no effect on the lethal action of rifampicin, itreduced fluoroquinolone lethality by 4- to 20-fold. Twofluoroquinolones, moxifloxacin and gatifloxacin, were equally lethalwith exponentially growing cells, but moxifloxacin was 2-fold moreactive during growth arrest.

Example.

The high potency of CEM-101 against Streptococcus pneumoniae,β-haemolytic and viridans group streptococci, Staphylococcus spp. andenterococci has been documented in early screening studies performedusing reference Clinical and Laboratory Standards Institute (CLSI)methods. Since mechanisms and occurrences of resistance are increasingrapidly that may compromise the MLSB-ketolide class, the post-antibioticeffects (PAE), bactericidal activity (MBC and killing curves) andpotential synergies of CEM-101 with five selected classes ofantimicrobial agents when testing wild type (WT) andphenotypically/genotypically defined resistant organism subsets wasassessed. MBC determinations for CEM-101, TEL, and CLR used CLSI methodsfor 40 strains (6 species groups). KC used 8 strains (6 species groups).PAE was tested (5 strains) at 4× concentration for 1 or 2 hoursexposure; TEL control. Drug interaction (synergy) studies were performedon 20 strains (7 S. aureus, 6β-haemolytic streptococci and 7 S.pneumoniae) by the checkerboard method. CEM-101 was combined with fiveagents (ceftriaxone, gentamicin, levofloxacin,trimethoprim/sulfamethoxazole [TMP/SMX] and vancomycin), eachrepresenting a distinct antimicrobial class.

The characterization of antimicrobial interactions into categories wasdefined as: complete synergy=four-fold or greater decrease in the MICvalues of both agents; partial synergy=four-fold or greater decrease inthe MIC value for one agent and a two-fold reduction in the MIC of theother; additive=twofold decrease in MIC values of both tested agents;antagonism=four-fold or greater increase in the MIC values of bothagents; and indifference=no decrease in the MIC values of either agentor only a two-fold decrease or increase in the MIC of one agent.

CEM-101 drug interaction (synergy) categories tested in combination withother antimicrobials. Synergy Indif- Antag- Indeter- Co-drug CompletePartial Additive ferent onism minate CRO 0 2 5 12 0 1 GEN 2 2 4 12 0 0LEV 0 0 3 17 0 0 TMP/SMX 0 2 4 14 0 0 VAN 0 1 6 13 0 0 All 2 7 22 68 0 1The most common interaction category observed for the CEM-101 drugcombination studies was indifference (68 occurrences), followed byadditive (22), and partial synergy (7) effects. Synergy was onlyobserved with CEM-101 and gentamicin for two S. pneumoniae strains. Thecombinations of CEM-101 with gentamicin, ceftriaxone, TMP/SMX,vancomycin and levofloxacin exhibited favorable interactions(complete/partial synergy or additive; 31% of all results) when testingS. pneumoniae strains; but indifferent interactions predominated amongtested S. aureus and S. pyogenes. None of the combinations evaluateddemonstrated antagonism.

MBC and killing curve studies: A total of 40 strains (10 S. pneumoniae,10 S. aureus, and 5 each of β-haemolytic streptococci, viridans groupstreptococci, coagulase-negative staphylococci [CoNS] and enterococci)were MIC tested followed by MBC determinations using CLSI procedures(MIC and MBC range, 0.008-16n/ml). The lowest concentration of a testedagent that killed ≧99.9% of the initial inoculum was defined as the MBCendpoint (Tables 2 and 3). Time kill bactericidal activity was performedfor CEM-101, TEL, CLR, and AZI on eight selected strains according tomethods described by Moody & Knapp, NCCLS M21-A3 and M26-A. Thecompounds were tested at 2×, 4×, 8×MIC; and colony counts were performedat T0, T2, T4, T8 and T24.

CEM-101 exhibited low MBC/MIC ratios (≦4) for BSA, SA andcoagulase-negative staphylococci; and 2-fold greater potency than TEL.SA, enterococci and some macrolide/CLN-resistant (R) strains had higherratios. KC results showed more rapid and greater cidal activity(concentration dependant) for CEM-101 compared to TEL. CEM-101/TEL PAE(hours) was: SA (2.3/2.6 hours), SPN (3.0/1.9), BSA (6.1/3.4), H.influenzae (3.7/1.2), M. catarrhalis (5.3/4.0). Interaction results withCEM-101 showing no antagonism and dominant additive or indifferenteffects. CEM-101 exhibited cidal activity against several Gram-positivespecies at rates and an extent greater than TEL. PAE for CEM-101 was2.3-6.1 and 3.7-5.3 hours for Gram-positive and Gram-negative strains,respectively. No antagonism was found in synergy analyses, with enhancedinhibition most noted for combinations with CRO, GEN and TMP/SMX.

Distribution of isolates according to MBC/MIC ratio for CEM-101, TEL,CLR and AZI No. of strains with Organism/Antimicrobial agent MBC/MICvalue of: (no. tested) 1 2 4 8 16 ≧32 S. pneumoniae (10) CEM-101 3 5 0 00 2 Telithromycin 2  6^(a) 0 0 0 2 Clarithromycin 2 3 1 0 0 —^(b)Azithromycin 2 4 0 0 0 —^(b) β-haemolytic streptococci (5) CEM-101 0 1 20 0 2 Telithromycin 0 1 1 1 0 2 Clarithromycin 0 0 1 1 0  2^(b)Azithromycin 0 0 0 0 2  2^(b) Viridans group streptococci (5) CEM-101 30 1 0 0 1 Telithromycin 2 1 1 0 0 1 Clarithromycin 0 0 1 0 0  3^(b)Azithromycin 0 0 0 0 1  3^(b) S. aureus (10) CEM-101 1 0 0 0 1 8Telithromycin 0 0 0 0 0 10  Clarithromycin 0 0 0 0 0  6^(b) Azithromycin0 0 0 0 0  6^(b) Coagulase-neg. staphylococci (5) CEM-101 1 1 0 3 0 0Telithromycin 0 0 0 0 2 3 Clarithromycin 0 0 0 0 0  4^(b) Azithromycin 00 0 0 0  4^(b) Enterococcus spp. (5) CEM-101 0 0 0 0 0 5 Telithromycin 00 0 0 0 5 Clarithromycin 0 0 0 0 0  2^(b) Azithromycin 0 0 0 0 0  2^(b)^(a)Includes six isolates with a MIC of ≦0.008 μg/ml and a MBC of 0.015μg/ml (off scale comparisons). ^(b)MBC was not evaluated on isolateswith resistant level MIC results.

CEM-101 showed rapid bactericidal activity (reduction of ≧3 log 10CFU/ml) against macrolide-susceptible strains of S. aureus, S.epidermidis, S. pneumoniae, S. pyogenes (only at 8×MIC) and viridansgroup streptococci, as well as a macrolide-resistant S. pyogenes.CEM-101 produced a greater reduction of CFU/ml and more rapid killingwhen compared to either TEL or the macrolides CLR and AZI.

Summary of Time Kill Curve Results

Organism Antimicrobial agent Antimicrobial activity S. aureus CEM-101Cidal at 2X, 4X, 8X (ATCC 29213) Telithromycin Cidal at 8X onlyClarithromycin Cidal at 8X only Azithromycin Cidal at 8X only S.epidermidis CEM-101 Cidal at 2X, 4X, 8X (095-2777A) Telithromycin StaticClarithromycin Static Azithromycin Static E. faecalis CEM-101 Static(ATCC 29212) Telithromycin Static Clarithromycin Static AzithromycinStatic S. pneumoniae CEM-101 Cidal at 2X, 4X, 8X (ATCC 49619)Telithromycin Cidal at 2X, 4X, 8X Clarithromycin Cidal at 2X, 4X, 8X(slow killing) Azithromycin Cidal at 2X, 4X, 8X (slow killing) S.pneumoniae CEM-101 Static (075-241B) Telithromycin Static S. pyogenesCEM-101 Cidal at 8X only (117-1612A) Telithromycin Cidal at 8X only(slow killing) Clarithromycin Cidal at 8X only (slow killing)Azithromycin Cidal at 8X only (slow killing) S. pyogenes CEM-101 Cidalat 2X, 4X, 8X (088-11708A) Telithromycin Cidal at 2X, 4X, 8X (slowkilling) S. mitis CEM-101 Cidal at 2X, 4X, 8X (112-1885A) TelithromycinCidal at 2X, 4X, 8X Clarithromycin Cidal at 8X only (slow killing)Azithromycin Cidal at 4X and 8X (slow killing)CEM-101 exhibited bactericidal activity when tested againstmacrolide-susceptible streptococci, CoNS and macrolide-resistantCLN-susceptible S. pneumoniae. CEM-101 MBC/MIC ratios can be high for S.aureus, but some strains showed MBC results remaining within thesusceptible range of concentrations.

PAE testing: PAE values for CEM-101 and TEL were determined usingestablished procedures which are consistent with those recommended byCraig and Gudmundsson. Both antimicrobial agents were tested againsteach isolate at 4× and 8× the MIC. Colony counts were performed atpre-antimicrobial exposure (T0) and after one or two hourspost-antimicrobial exposure (T1 or T2). After “diluting out” theantimicrobial agents (1:1000), colony counts were performed every houruntil turbidity was noted (up to 10 hours post dilution) to determinethe length of PAE. The tested Gram-positive and Gram-negative pathogenswere as follows: S. aureus ATCC 29213; H. influenzae ATCC 49247; S.pneumoniae ATCC 49619; S. pyogenes WT (177-1612A); and M. catarrhalis WT(117-10142A).

PAE results for CEM-101 compared to TEL measured in hours. AntimicrobialS. aureus S. pneumoniae S. pyogenes H. influenzae M. catarrhalisconcentration ATCC 29213 ATCC 49619 117-1612A ATCC 49247 117-10142ACEM-101 (4X MIC) 2.3 3.0 6.1 3.2 6.3 Telithromycin 2.6 1.9 3.4 1.2 4.0(4X MIC) Exposure (hours) 2 1 2 1 2After two hours of exposure, the PAE of CEM-101 (2.3 hours) was similarto TEL (2.6 hours) when tested against S. aureus at 4×MIC value. Byincreasing the concentration during the exposure to 8× the MIC, CEM-101PAE was extended to 3.9 hours (data not shown). CEM-101 PAE testedagainst S. pneumoniae and S. pyogenes was 3.0 and 6.1 hours compared to1.9 and 3.4 hours, respectively for TEL. CEM-101 PAE againstGram-negative pathogens also favored the new agent versus the olderketolide.

The compounds described herein show a significant concentration andexposure-dependent PAE against Gram-positive, as evidenced by CEM-101(average PAE, 3.8 hours) and Gram-negative (average PAE, 4.5 hours)pathogens commonly associated with CA-RTI and uSSSI. Overall, the PAE ofCEM-101 was longer than that of TEL.

Example.

Activity on Chlamydia. CEM-101, TEL, AZI, CLR, and doxycycline wereprovided as powders and solubilized according to the instructions of themanufacturers. Drug suspensions were made fresh each time the assay wasrun.

C. pneumoniae: Isolates of C. pneumoniae tested included a referencestrain (TW 183), 9 isolates from children and adults with pneumonia fromthe United States (AR39, T2023, T2043, W6805, CWL 029, CM-1), an isolatefrom a child with pneumonia from Japan (J-21), and 2 strains frombronchoalveolar lavage specimens from patients with humanimmunodeficiency virus infection and pneumonia from the United States(BAL15 and BAL16).

C. trachomatis: 10 isolates of C. trachomatis, including standardisolates from the ATCC (E-BOUR, F-IC-CAL3, C-HAR32, J-UW-36, L2434,D-UW-57kx, B-HAR-36) and recent clinical isolates (N18(cervical),N19(cervical), 7015(infant eye))

In vitro susceptibility testing: Susceptibility testing of C. pneumoniaeand C. trachomatis was performed in cell culture using HEp-2 cells grownin 96-well microtiter plates. Each well was inoculated with 0.1 ml ofthe test strain diluted to yield 10³ to 10⁴ IFU/per ml, centrifuged at1,700×g for 1 hr. and incubated at 35° C. for 1 hr. Wells were aspiratedand overlaid with 0.2 mL of medium containing 1 μg of cycloheximide permL and serial two-fold dilutions of the test drug.

Duplicate plates were inoculated. After incubation at 35° C. for 48-72hrs, cultures were fixed and stained for inclusions withfluorescein-conjugated antibody to the lipopolysaccharide genus antigen(Pathfinder, Kallestad Diagnostics, Chaska, Minn.). The minimalinhibitory concentration (MIC) is the lowest antibiotic concentration atwhich no inclusions were seen. The minimal bactericidal concentration(MBC) was determined by aspirating the antibiotic containing medium,washing wells twice with phosphate buffered saline and addingantibiotic-free medium. Cultures were frozen at −70° C., thawed, passedonto new cells, incubated for 72 hrs then fixed and stained as above.The MBC is the lowest antibiotic concentration that results in noinclusions after passage. All tests were run in triplicate.

Activities of CEM-101 and other antibiotics against 10 isolates of C.pneumoniae MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 1010.25-1.0  0.25 0.25 0.25-1.0  0.25 Telithromycin 0.015-0.25  0.06 0.060.015-0.25  0.06 Azithromycin 0.015-0.125 0.125 0.125 0.015-0.125 0.125Clarithromycin 0.015-0.125 0.06 0.06 0.015-0.125 0.06 Doxycycline0.015-0.06  0.06 0.06 0.015-0.06  0.06

Activities of CEM-101 and other antibiotics against 10 isolates of C.trachomatis MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 1010.125-0.5  0.25 0.25 0.125-0.5  0.25 Telithromycin 0.015-0.25  0.06 0.060.015-0.25  0.06 Azithromycin 0.015-0.125 0.125 0.125 0.015-0.125 0.125Clarithromycin 0.015-0.125 0.06 0.06 0.015-0.125 0.06 Doxycycline0.015-0.06  0.06 0.06 0.015-0.06  0.06The results of this study demonstrated that CEM-101 has in vitroactivity against C. trachomatis and C. pneumoniae comparable to othermacrolides and ketolides.

Example.

Tissue distribution. CEM-101 was well absorbed and distributed to thetissue. In the rat at 250 mg/kg/d, mean lung and liver concentrations ofCEM-101 were 17 and 15-fold higher than in plasma. Lung and liverconcentrations were 503 and 711-fold higher than plasma concentrationsat the 200 mg/kg/d dose in monkeys. Concentrations of CEM-101 in theheart were significantly lower than levels found in lung or liver withlevels 5 and 54-fold higher than plasma concentrations in rat andmonkey, respectively.

Example.

Activity of CEM-101 Against Invasive Isolates of N. meningitidis (NM)from a worldwide collection. Colonization of the nasopharynx (NP) by NMcan lead to invasive meningococcal disease. Chemoprophylaxis is used toeradicate NP colonization and prevent transmission to nonimmunecontacts. Described herein is the determination of the activity ofCEM-101 tested against invasive clinical isolates of NM. 62 isolates(91.9% blood culture) of NM were collected from 29 medical centers inNorth and South America and Europe (1997-2008). Strains were tested forsusceptibility (S) to CEM-101 and 10 comparators including β-lactams,fluoroquinolones (FQs), macrolides and three other classes by CLSI brothmicrodilution methods. Serological identification was performed forserogroups (SGs) B, C, Y and W135.

Susceptibility to penicillin was 82.3% with no resistant (R) strainsdetected. All isolates were susceptible to ceftriaxone, AZI, minocyclineand rifampin. Isolates were susceptible to FQs (≦0.015 μg/ml); however,13 strains had reduced susceptibility to nalidixic acid (MIC≧8 μg/ml),which may correlate with diminished susceptibility to FQs. Resistance totrimethoprim/sulfamethoxazole (T/S) was 59.7%. Among the MLS_(B) classagents, CEM-101 was the most active (MIC₉₀, ≦0.015 μg/ml) compared toTEL (0.03 μg/ml), AZI and CLR (0.12 μg/ml) and erythromycin (0.25μg/ml). The prevalence rates (%) of SGs were C (41.7), B (38.3), Y(16.7) and W135 (3.3). CEM-101 was the most active compound testedagainst MLS_(B) NM strains (all MICs, ≦0.06 μg/ml) with a potency≧2- to32-fold greater than other class agents. CEM-101 was active against NMisolates non-S to β-lactams and T/S.

Antimicrobial MIC (μg/ml) % agent 50% 90% Range Susc.^(a) % Res.^(a)CEM-101 ≦0.015 ≦0.015 ≦0.015-0.06  —^(b) — TEL ≦0.015 0.03 ≦0.015-0.12 —— AZI 0.06 0.12 ≦0.015-0.12 100.0 — CLR 0.03 0.12 ≦0.015-0.25 — —Erythromycin 0.12 0.25    0.03-0.5 — — Penicillin 0.03 0.12 ≦0.015-0.25  82.3 0.0 Ceftriaxone ≦0.015 ≦0.015 ≦0.015 100.0 — Ciprofloxacin ≦0.008≦0.008 ≦0.008-0.015 100.0 0.0 Levofloxacin ≦0.008 ≦0.008 ≦0.008-0.015100.0 0.0 Minocycline 0.12 0.12 ≦0.008-0.25  100.0 — Rifampin 0.015 0.06≦0.008-0.12  100.0 0.0 Trimethoprim/ 0.5 2 ≦0.06-4    37.1 59.7 sulfamethoxazole ^(a)Susceptibility criteria based upon the CLSI(M100-S19, 2009). ^(b)No susceptibility criteria have been proposed, butAZI at ≦2 μg/ml is considered susceptible.

Example.

Ability of CEM-101 to Select Resistant S. pyogenes Clones by MultistepMethod. It has been reported that S. pyogenes retain β-lactamsusceptibility but are sometimes macrolide resistant. TEL is activeagainst all macrolide resistant S. pyogenes genotypes except for erm(B).It has been discovered herein that CEM is 2 to 4-fold more active thanTEL. Described herein is the tested capability of CEM, AZI, CLR, TEL,and CLN to select resistant clones of S. pyogenes in 5 strains withvarying resistotypes.

One strain each was tested as follows: macrolide susceptible, erm(B),mef(A), erm(A), L4 ribosomal protein mutation. CLSI macrodilution wasused for MIC tests. Serial passages were daily in MHB+5% lysed horseblood for each strain at subinhibitory drug concentrations, taking foreach subsequent passage an inoculum from the tube 1-2 dilutions belowthe MIC that matched turbidity of a growth control. Daily passages werecontinued until the MIC increased >4-fold (max. 50 passages). Resistantclones were subcultured 10× in drug-free medium to test stability ofselected resistance. Identity between parents and resistant clones wasconfirmed by PFGE and macrolide resistant phenotypes identified by PCR.

Parental MICs (μg/ml) were: CEM-101, 0.008-1; AZI, 0.06-4; CLR, 0.03-4;TEL, 0.03-8; and CLN, 0.06 (1 strain with AZI, CLR, CLN MICs>64 μg/mlwas not tested). CEM-101 MICs increased after 18-43 days in 3/5 strains,rising from 0.03-1 μg/ml (parents) 0.25-8 μg/ml (resistant clones). MICsfor 2 of the clones did not go above 0.25 μg/ml when passages werecontinued for the maximum 50 days. AZI had resistant clones after 5-35days in 3/4 strains tested, with MICs rising from 0.06-4 μg/ml(parents)→1->64 μg/ml (R clones). CLR had resistant clones after 6 daysin 1/4 strains tested, with MICs rising from 0.5 mg/ml (parent)→>64μg/ml (resistant clone). TEL had resistant clones after 6-22 days in 2/4strains tested, with MICs rising from 0.03-8 μg/ml (parents)→0.25->64μg/ml (resistant clones), CLN had resistant clones after 34-43 days in2/3 strains tested with MICs rising from 0.06 μg/ml (parents)→0.5->64μg/ml (resistant clones). In 2 of the 3 resistant clones with CEM-101[parents erm(A), L4], MICs were 0.25 μg/ml and only in the 1 strain witherm(B) did CEM-101 MICs rise from 1-8 μg/ml.

Example.

Capability of CEM-101 to Select for Resistant (R) Pneumococcal Clones byMultistep Method. Drug resistant strains of S. pneumoniae occurworldwide. It has been discovered herein that CEM-101 is 2 to 4-foldmore active than TEL against macrolide resistant pneumococci. Describedherein is the tested ability of CEM to select for resistant clones of S.pneumoniae compared to AZI, CLR, TEL, CLN in 8 pneumococcal strains withvarying resistotypes.

One strain in each of the following was tested: macrolide susceptible,erm(B), mef(A), ermB+mefA, erm(A), L4, L22, and 23S rRNA ribosomalprotein mutations. CLSI macrodilution was used for MIC testing. Serialpassages were daily in MHB+5% lysed horse blood for each strain atsubinhibitory drug concentrations, taking for each subsequent passage aninoculum from the tube 1-2 dilutions<MIC that matched turbidity of agrowth control. Daily passages were continued until the MICincreased >4-fold (min. 14, max. 50 passages). Resistant clones weresubcultured 10× in drug-free medium to test stability of selectedresistance. Identity between parents and resistant clones was confirmedby PFGE and macrolide resistant phenotypes identified by PCR.

Parental MICs (μg/ml) were: CEM-101, 0.004-1; AZI, 0.03-8; CLA,0.016-16; TEL, 0.004-0.5; CLI, 0.016-1 (four strains with AZI, 2 CLR, 2CLN MICs>64 μg/ml were not tested). CEM-101 MICs increased after 14-43days in all 8 strains tested. For 7 strains, MICs rose from 0.004-0.03μg/ml (parents)→>0.06-0.5 μg/ml (resistant clones) in 14-43 days. Forthe eighth strain, containing erm(B)+mef(A), MICs rose from 1 μg/ml(parent)→32 μg/ml (resistant clone) in 18 days. AZI had resistant clonesafter 14-29 days in 3/4 strains with MICs rising from 0.03-2 μg/ml(parents)→0.5->64 μg/ml (resistant clones). CLR had resistant clonesafter 14-49 days in 5/6 strains with MICs rising from 0.03-16 μg/ml(parents)→16->64 μg/ml (resistant clones). TEL had resistant clonesafter 14-38 days in 5/8 strains with MICs rising from 0.004-0.5 μg/ml(parents) to 0.06->64 μg/ml (resistant clones). CLN had resistant clonesafter 14-43 days in 2/5 strains with MICs rising from 0.03-0.06 μg/ml(parents)→0.25->64 μg/ml (resistant clones). CEM-101 yielded clones withhigher MICs in all 8 strains, but 7 of 8 strains had clones with CEM-101MICs≦0.5 μg/ml and only in the 1 strain with erm(B)+mef(A) with aparental MIC=1 μg/ml was a resistant clone with an MIC=32 μg/ml found.

Example.

Human THP-1 macrophages were used. Accumulation was measured bymicrobiological assay. Intracellular activity was determined againstphagocytized S. aureus (ATCC 25923; MICs: CEM-101, 0.125 mg/L; AZI, 0.5mg/L) using a dose-response approach (AAC 2006; 50:841-51). Verapamil(100 μM) and gemfibrozil (250 μM) were used as inhibitors ofP-glycoprotein and MRP, respectively (AAC, 2007; 51:2748-57).

Accumulations and activities after 24 h incubation, with and withoutefflux transporters inhibitors, are shown in the following Table, whereCc/Ce is the apparent cellular to extracellular concentration ratio, andE_(max) is the maximal decrease of intracellular cfu compared topost-phagocytosis inoculum (calculated from non-linear regression[sigmoidal] of dose-effect response experiments).

AZI CEM-101 Intracellular activity Intracellular activity (Δ log cfu at24 h) (Δ log cfu at 24 h) Static dose Cc/Ce¹ Static dose ConditionCc/Ce¹ (24 h) (mg/L) E_(max) ² (24 h) (mg/L) E_(max) ² control  127.7 ±23.5 ~7.0   0.10 ± 0.09 268.1 ± 7.1  ~0.02 −0.85 ± 0.23^((b)) Verapamil216.37 ± 46.6^((a)) ~0.2 −0.37 ± 0.15 290.2 ± 12.9 ~0.03 −0.59 ±0.22^((b)) Gemfibrozil 129.12 ± 2.69 ~3.8 −0.12 ± 0.20 308.2 ± 47.8~0.03 −0.73 ± 0.20^((b)) ^((a))Statistically significant from bothcontrol and Gemfibrozil; ^((b))not statistically significant.

Example.

Intracellular activity of antibiotics. The determination of antibioticactivity against intraphagocytic S. aureus strain ATCC 25923 wasdetermined. Full dose-responses studies were performed to assess theimpact of active efflux in the modulation of the intracellular activityof CEM-101 and AZI against intraphagocytic S. aureus (strain ATCC 25923[MICs: CEM-101, 0.125 mg/L; AZI, 0.5 mg/L]. Antibiotics were compared at24 h for: (i) their relative static concentration (Cs), and (ii) theirrelative maximal efficacy (E). While verapamil (but not gemfibrozil)increases the intracellular activity of AZI, neither inhibitor havesignificant effect on the activity of CEM-101, suggesting that thelatter, in contrast with AZI, is not a substrate of the correspondingeukaryotic transporters.

Example.

Cellular accumulation of antibiotics. The cellular content in macrolideswas measured in THP-1 macrophages by microbiological assay, using S.aureus ATCC 25923 as test organism. Cell proteins was assayed inparallel using the Folin-Ciocalteu/Biuret method. The cell associatedcontent in macrolides was expressed by reference to the total cellprotein content, and converted into apparent concentrations using aconversion factor of 5 μL per mg of cell protein (as commonly used forcultured cells).

The cellular accumulation of CEM-101 in comparison with that of AZI inTHP-1 cells was first measured FIG. 5 (panel A). At 24 h, bothantibiotics concentrate to large extents in cells, but with a largervalue (Cc/Ce) for CEM-101. In a second stage, whether CEM-101 is asubstrate of Pgp or MRP efflux transporters was investigated FIG. 5(panel B). Using a Pgp (verapamil) or MRPs inhibitor (gemfibrozil), nosignificant variations of the cellular accumulation of CEM-101 areobserved while verapamil increases significantly the cellularaccumulation of AZI.

Uptake of CEM-101 was linear over time, reaching accumulation levelsabout 375-fold within 24 h (AZI, 160X, CLR, 30X, TEL, 21X). Accumulationwas suppressed by acid pH or addition of the proton ionophore monensin,but not modified by verapamil or gemfibrozil (preferential inhibitors ofPgp and MRP, respectively). Panel B shows that the accumulation of bothCEM-101 and AZI was reduced when the experiments were conducted atacidic pH, with the change occurring almost entirely when the pH wasbrought from 7 to 6. Panel C shows that monensin, which is known todecrease the cellular accumulation of many weak organic bases, alsoalmost completely suppressed the accumulation of both CEM-101 and AZI.In contrast, verapamil, an inhibitor of the P-glycoprotein effluxtransporter (Pgp, also known as MDR1), increased the accumulation of AZIwithout affecting that of CEM-101, whereas gemfibrozil, an inhibitor ofmultidrug resistance proteins (MRP) and other organic anion transportersdid not affect either compound. Neither verapamil nor gemfibrozilaffected the accumulation of TEL or CLR (data not shown). The efflux ofCEM-101 from cells incubated with 10 mg/L of CEM-101 for 1 h and thentransferred into drug-free medium was examined. Efflux proceeded in abimodal fashion, with half of the cell-associated drug being releasedwithin approximately 10 min, followed by a slower release phase ofseveral hours (data not shown).

Example.

Macrolides accumulate in eukaryotic cells and are consideredadvantageous for the treatment of intracellular infections. Ketolidesare active against erythromycin-resistant organisms. The cellularaccumulation and intracellular activity of CEM-101 towards theintracellular forms of Staphylococcus aureus (S. a.), Listeriamonocytogenes (L. m.), and Legionella pneumophila (L. p.) in comparisonwith AZI, CLR, and TEL is shown in the following table.

MIC^(a) Cs^(b) E_(max) ^(c) CEM-101 S.a. 0.06 0.022 −0.86 L.m. 0.0040.11 −0.66 L.p. 0.004 0.018 −1.03 AZI S.a. 0.5 >50 0.04 L.m. 1 11.6−0.81 L.p. 0.016 2.90 −0.83 CLR S.a. 0.5 0.84 −0.18 L.m. L.p. 0.007 0.12−0.71 TEL S.a. 0.25 0.63 −0.29 L.m. L.p. 0.007 0.06 −0.63 ^(a)mg/L;^(b)static concentration (mg/L) at 24 h; ^(c)Δ log₁₀ CFU at 24 hcompared to the post-phagocytosis inoculum

Example.

MICs and extracellular activities of antibiotics were determined in MHBat both neutral and acidic pH. Intracellular activity was determinedagainst S. aureus (ATCC 25923) phagocytosed by THP-1 macrophages aspreviously described (AAC, 2006, 50:841-851), Results were expressed asa change of efficacy compared to time 0 h.

Conditions CEM-101 AZI CLR TEL MICs (mg/L) (i) pH 7.4 0.125 0.5 0.5 0.5(ii) pH 5.5  1-2 256 16 8 Extracellular activity (24 h): Δ log cfu fromtime 0 h (i) Broth pH 7.4 Emax¹ −1.4 ± 0.1 −1.2 ± 0.6 −1.4 ± 0.2 −1.0 ±0.4 Static ~0.06 ~3.63 ~1.41 ~0.28 dose² R² 0.964 0.860 0.965 0.868 (ii)Broth pH 5.5 Emax¹ −1.6 ± 0.4 +2.1 ± 0.1 −1.5 ± 0.8 −1.4 ± 0.9 Static~1.48 / ~10.47 ~9.33 dose² R² 0.915 / 0.911 0.879 Intracellular activity(24 h): Δ log cfu from time 0 h Emax¹ −0.8 ± 0.2 0.10 ± 0.0 −0.1 ± 0.1−0.4 ± 0.2 Static ~0.02 ~7.8 ~0.98 ~0.23 dose² R² 0.906 0.980 0.9740.935 THP-1 Emax¹ −0.8 ± 0.2  0.1 ± 0.1 −0.1 ± 0.1 −0.4 ± 0.1 Static~0.02 ~10 ~0.98 ~0.28 dose² ¹Maximal decrease of intracellular cfucompared to initial, post-phagocytosis inoculum (calculated fromnon-linear regression [sigmoidal] of dose-effect response) run in broth(extracell.) or with infected macrophages (intracell.) ²Extracellularconcentration (Cs in mg/L) yielding an apparent static effect.Comparative pharmacological descriptors (Emax and static concentrations[Cs]) obtained from the dose-responses studies. Dose-response studies inMueller-Hinton broth. Against S. aureus ATCC 25923 and in broth, at pH7.4, CEM-101 is systematically more active than AZI, CLR and TEL; at pH5.5, AZI, CLR and TEL show significant decrease of their potencies,while CEM-101 shows less change.Compared to AZI, CLR and TEL, CEM-101 activity was less affected byacidic pH of the broth and showed greater potency (lower static dose)and larger maximal efficacy (Emax) against intracellular S. aureus.

Example.

Cell lines. Experiments were performed with THP-1 cells (ATCC TIB-202;American Tissue Culture Collection, Manassas, Va.), a humanmyelomonocytic cell line displaying macrophage-like activity (see, e.g.,Barcia-Macay et al., Antimicrob. Agents Chemother. 50:841-851 (2006)).Assay of the cell-associated macrolides and calculation of the apparentcellular-to-extracellular-concentration ratios. Macrolides were assayedby a microbiological method, using S. aureus ATCC 25923 as a testorganism. Cell proteins were measured in parallel using theFolin-Ciocalteu/biuret method. The cell-associated contents inmacrolides were expressed by reference to the total cell protein contentand converted into apparent concentrations using a conversion factor of5 μL per mg of cell protein, an average value found for many culturedcells.

Bacterial strains, susceptibility testing, and 24-h dose-response curvestudies with broth. S. aureus ATCC 25923 (methicillin [meticillin]sensitive), L. monocytogenes strain EGD, and L. pneumophila strain ATCC33153 were used in the present study. MIC determinations were performedin Mueller-Hinton broth (for S. aureus) and tryptic soy broth (for L.monocytogenes) after a 24-h incubation, or in α-ketoglutarate-bufferedyeast extract broth (for L. pneumophila) after a 48-h incubation. For S.aureus studies, 24-h concentration-response experiments in acellularmedium were performed in Mueller-Hinton broth.

Cell infection and assessment of antibiotic intracellular activities.Infection of THP-1 cells and assessment of the intracellular activity ofantibiotics were performed using conventional methods for S. aureus andL. monocytogenes or with minor adaptations for L. pneumophila using (i)a multiplicity of infection of 10 bacteria per macrophage and (ii)gentamicin (50 mg/liter) for 30 to 45 min for the elimination ofnonphagocytosed bacteria.

Statistical analyses. Curve-fitting statistical analyses were performedwith GraphPad Prism version 4.03 and GraphPad Instat version 3.06(GraphPad Software, San Diego, Calif.).

Example.

Susceptibility toward S. aureus ATCC 25923, Listeria monocytogenes EGD,and Legionella pneumophila ATCC 33153. CEM-101 showed lower MICs thanAZI against the three selected organisms (S. aureus, 0.06 and 0.5mg/liter; L. monocytogenes, 0.004 and 1 mg/liter; and L. pneumophila,0.004 and 0.016 mg/liter) in conventional susceptibility testing. TheMICs of CEM-101, TEL, AZI, and CLR against S. aureus and L.monocytogenes were measured in broths adjusted to pH values ranging from5.5 to 7.4. The range was selected to cover the values at which theantibiotics could be exposed in the extracellular milieu orintracellularly for the two organisms considered. As illustrated in FIG.1, all four drugs showed a marked decrease in potency against bothorganisms when the pH was decreased from 7.4 to 5.5, with AZIdemonstrating the most significant loss of activity. CEM-101 retainedthe most activity, consistently showing the lowest MICs throughout theentire pH range investigated, with values (mg/liter) ranging from 0.06(pH 7.4) to 0.5 (pH 5.5) for S. aureus (ATCC 25923) and 0.0039 (pH 7.4)to 0.25 (pH 5.5) for L. monocytogenes (EDG). For L. pneumophila (datanot shown), the MIC of CEM-101 increased from 0.005 to 0.01 and that ofAZI from approximately 0.01 to 0.25 mg/liter when the pH of the brothwas decreased from 7.4 to 6.5 (no determination could be made at lowerpH values because of absence of growth).

Example.

Time and concentration effects against extracellular and intraphagocyticS. aureus. Short-term (6-h) time-kill curves were obtained for CEM-101in comparison with those for AZI against S, aureus (ATCC 25923) in brothand after phagocytosis by THP-1 macrophages using two single fixedconcentrations of 0.7 and 4 mg/liter. The lower concentration was chosento be relevant to the serum concentration of AZI and CEM-101, and thehigher concentration was selected to be above the MIC of AZI for theorganisms of interest. Results presented in FIG. 3 show that under theseconditions, only CEM-101 was able to significantly decrease CFU in brothas well as in THP-1 macrophages at the 0.7-mg/liter concentration. Atthe 4-mg/liter concentration in broth, AZI eventually achieved the sameantibacterial effect as CEM-101, but at a lower rate (5 h compared to 1h). In THP-1 macrophages, no consistent activity was detected for AZI,even at the 4-mg/liter concentration, whereas CEM-101 again achieved areduction of approximately 1.5 log 10 CFU, similar to the magnitude seenat the 0.7-mg/liter concentration. In all situations with CEM-101, themaximal decrease of CFU was obtained within 1 h and was maintainedthereafter.

We then performed concentration-response experiments at a fixed timepoint (24 h) to obtain the pertinent pharmacological descriptors ofCEM-101 activity (relative potency [50% effective concentration {EC50}],apparent static concentration [C₈], and relative maximal efficacy[E_(max)] in comparison with CLR, AZI and TEL activity (additionaldetails are described in Barcia-Macay et al., Pharmacodynamic evaluationof the intracellular activities of antibiotics against Staphylococcusaureus in a model of THP-1 macrophages Antimicrob. Agents Chemother.50:841-851 (2006)). Data are presented in FIG. 2 as a function of (i)weight concentrations (mg/liter) and (ii) multiples of the MICs (asdetermined in broth at pH 7.4). The numerical values of thecorresponding pharmacological descriptors are shown in the Table.Pertinent regression parameters' (with confidence intervals [CI]), andstatistical analysis of the dose-response curves illustrated in FIG. 2.

broth⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄) (Cl) C_(S) ^(⋄⋄) R²CEM-101 −1.37 mg/L 0.03 0.06 0.973 (−1.67 to −1.08) (0.02 to 0.06) a; Aa; A ×MIC 0.48 0.88 (0.26 to 0.91) a; A TEL −1.00 mg/L 0.12 0.29 0.892(−1.78 to −0.22) (0.03 to 0.52) a; A b; A ×MIC 0.46 0.96 (0.11 to 2.06)a; A AZI −1.23 mg/L 1.78 3.4 0.872 (−2.55 to 0.083) (0.45 to 7.02) a; Ac; A ×MIC 3.55 6.87 (0.90 to 14.0) b; A CLR −1.41 mg/L 0.80 1.32 0.956(−1.95 to −0.87) (0.41 to 1.56) a; A c; A ×MIC 1.59 2.65 (0.81 to 3.1) a, b; A anti- THP-1 macrophages⁺⁺ R² biotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄)(CI) C₅ ^(⋄⋄) (CI) CEM- −0.86 mg/L 0.0068 0.022 0.927 101 (−1.36 to(0.0023 to 0.020)  −0.37) a; B a; B ×MIC 0.11 0.35 (0.037 to 0.32) a; BTEL −0.29 mg/L 0.024 0.63 0.954 (−0.70 to 0.12) (0.007 to 0.088) b; B b;B ×MIC 0.097 1.04 0.027 to 0.35 a; B AZI  0.04 mg/L 0.11 >50 0.983(−0.23 to 0.32) (0.05 to 0.22) b; B c; B ×MIC 0.22 >100 0.11 to 0.45 a;B CLR −0.18 mg/L 0.046 0.84 0.974 (−0.52 to 0.16) (0.018 to 0.12)  b; Bb, c; B ×MIC 0.093 1.68 0.035 to 0.25 a; B ^(a)using all data pointsshown in FIG. 4 (data from samples without antibiotic when theextracellular concentration of an antibiotic is lower than 0.01 ×MIC (5)⁺original inoculum [time = 0 h]: 0.97 ± 0.24 × 10⁶ CFU/mL (n = 3)⁺⁺original (post-phagocytosis) inoculum [time = 0 h]: 2.74 ± 0.55 × 10⁶CFU/mg protein (n = 3) ^(♦)CFU decrease (in log₁₀ units) at time = 24 hfrom the corresponding original inoculum, as extrapolated for antibioticconcentration = ∞; samples yielding less than 5 counts were consideredbelow detection level. ^(⋄)concentration (in mg/L or in ×MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1); ^(⋄⋄)concentration (in mg/L or in ×MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation; StatisticalAnalyses. Analysis of the differences between antibiotics (per columnfor the corresponding rows; one-way ANOVA with Tuckey test for multiplecomparisons between each parameter for all drugs): figures withdifferent lower case letters are significantly different from each other(p < 0.05). Analysis of the differences between broth and THP-1macrophages (per row for the corresponding columns; unpaired, two-tailedt-test): figures with different upper case letters are significantlydifferent from each other (p < 0.05).

The activities in both broth and THP-1 macrophages developed in aconcentration-dependent fashion, as denoted by the sigmoidal shape ofeach best-fit function (Hill equation). In broth, the relative efficacyof CEM-101 (E_(max) of −1.37 log₁₀) was similar to that of the otherdrugs (E_(max) values of −1.00 to −1.41 log₁₀). In THP-1 macrophages,the relative efficacy of CEM-101 was significantly decreased compared tothat in broth of −0.86 log₁₀), but not to the same extent as those ofthe other drugs, which essentially became bacteriostatic only (E_(max)values of 0.04 to −0.29 log₁₀). On a weight basis, CEM-101 had higherrelative potencies (lower E₅₀ values) and lower static concentrations(lower C_(s) values) than all three comparator drugs in both broth andin THP-1 macrophages. When the data were analyzed as a function ofequipotent concentration (multiples of the MIC), these differences inEC₅₀ values were reduced, indicating that the MIC was the main drivingparameter in this context. In broth, even when analyzed as multiples ofthe MIC, CEM-101 and CLR still showed significantly lower EC₅₀s than TELand AZI.

Example.

Activity against intraphagoctic L. monocytogenes and L. pneumophila. Thesame approach was used as that for S. aureus to assess the activities ofCEM-101 and AZI against phagocytized L. monocytogenes and L. pneumophilato obtain information on concentration-effect relationships and on thecorresponding pertinent pharmacological descriptors. As shown in FIG. 4,a relationship compatible with the Hill equation was observed in allcases, although the limited growth of L. pneumophila made the fitting offunctions somewhat more uncertain. When the data were plotted againstweight concentration, it appeared that CEM-101 had a higher relativepotency (lower EC50) than AZI for both L. monocytogenes and L.pneumophila. This difference was reduced but nevertheless remainedsignificant when data for L. pneumophila were plotted against multiplesof the MIC, indicating that the MIC was an important but not theexclusive driver of intracellular activity against this organism.Conversely, no difference in the responses was seen for L. monocytogeneswhen data were expressed as multiples of the MIC. Numerical values ofthe pertinent pharmacological descriptors and statistical analysis oftheir differences are shown in the Table.

Pertinent regression parameters^(a) (with confidence intervals [C]), andstatistical analysis of the dose-response curves illustrated in FIG. 4.

anti- L. monocytogenes EGD⁺ biotic E^(max♦) (CI) EC₅₀ ^(⋄) (Cl) C_(S)^(⋄⋄) R² −0.66 mg/L 0.020 0.11 0.934 CEM- (−1.28 to −0.037) (0.005 to0.073) 101 a a ×MIC 5.00 0.88 (1.36 to 18.5) a AZI −0.81 mg/L 2.66 11.60.953 (−2.11 to 0.48)  (0.91 to 7.73) a b ×MIC 2.66 11.6 (0.81 to 3.1) a L. pneumophila anti- ATCC 33153⁺⁺ biotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄)(CI) C_(S) ^(⋄⋄) R² −1.03 mg/L 0.052 0.018 0.920 CEM- (−1.34 to −0.72)(0.012 to 0.23)  101 a a ×MIC 13.1 4.56 (3.02 to 57.0) a AZI −0.83 mg/L2.86 2.90 0.903 (−2.00 to 0.34)  (0.17 to 48.6) a b ×MIC 179.0 181 (10.5 to 3038) b ^(a)using all data points shown in FIG. 4 (data fromsamples without antibiotics were not used because of evidence ofextracellular growth when the extracellular concentration of anantibiotic is lower than 0.01 ×MIC (5). ⁺original (post-phagocytosis)inoculum [time = O h; CFU/mg protein]: L. monocytogenes, 1.67 ± 0.22 ×10⁶ (n = 3); L. pneumophila, 0.94 ± 0.60 × 10⁶. ^(♦)CFU decrease (inlogo units) at time = 24 h (L. monocytogenes) or 48 h (L. pneumophila)from the corresponding original inoculum, as extrapolated for antibioticconcentration ∞; samples yielding less than 5 counts were consideredbelow detection level. ^(⋄)concentration (in mg/L or in ×MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1). ^(⋄⋄)concentration (in mg/L or in ×MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation. Statisticalanalyses: analysis of the differences between the two antibiotics (percolumn for the corresponding rows; unpaired, two-tailed t-test): figureswith different lower case letters are significantly different from eachother (p < 0.05).

Example.

Dose-response studies in infected THP-1 macrophages Againstintraphagocytic S. aureus ATCC 25923, CEM-101 is more potent than AZI,CLR and TEL (lower Cs), In addition, CEM-101 is able to reduce theintracellular inoculum (E_(max)˜1 log), which is not observed with anyof AZI, CLR and TEL.

CEM-101 uptake within cells (ii): role of the cell type MDCKsur-expressing THP-1 J774 MDCK the MDR1 (human (murine (canine effluxCells macrophages) macrophages) epith. cells) transporters Cc/Ce ~50-150~60 ~45 ~30 at 5 h

Example.

Example Dose-response studies of CEM-101 vs. comparators (AZI, CLR andTEL) against intracellular S. aureus ATCC 25923 (THP-1 macrophages). SeeFIG. 7 and the Table.

CEM-101 AZI CLR TEL Emax −0.80 ± 0.11 0.04 ± 0.11 −0.18 ± 0.13 −0.29 ±0.16 Cs (mg/L) ~0.01 >50 ~0.86 ~0.27

Example.

Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages were measured. See FIG. 6 bars represent theMICs (in mg/L) or the extracellular static dose.

METHOD. Mouse peritoneal macrophages were infected with viable M.leprae, the drugs are added and incubated at 33° C. for 3 days. After 3days macrophages were lysed to release the intracellular M. leprae whichwere then assayed for viability by radiorespirometry and viabilitystaining. CEM-101 shows efficacy against intracellular M. lepraeviability.

The Thai-53 isolate of M. leprae, maintained by serial passages inathymic nu/nu mice footpads, was used for all experiments. For axenictesting freshly harvested viable M. leprae were incubated in mediumalong with different concentrations of the drugs (CEM-101, CLR andrifampin) for 7 days at 33° C. At the end of this incubationdrug-treated M. leprae were subjected to radiorespirometry to assessviability based on oxidation of palmitate and staining with viabilitydyes to assess the extent of membrane damage. For intracellular testingperitoneal macrophages from Swiss mice were infected with freshlyharvested viable M. leprae at an MOI of 20:1 for 12 hours. At the end ofthe infection extracellular bacteria were washed and drugs added atdifferent concentrations and incubated for 3 days at 33° C. At the endof 3 days cells were lysed to obtain the intracellular M. leprae forradiorespirometry and viability staining.

CEM-101 at 0.15 μg/ml was able to significantly (P<0.001) reduce theviability of M. leprae in both axenic and intracellular cultures whencompared to controls. Inhibition by CEM-101 was not statisticallydifferent from inhibition obtained with CLR under identical conditionsand at the same concentration.

Example.

Emerging Telithromycin-Resistant β-Haemolytic Streptococci (BHS).CEM-101 was tested against a collection of 43 TEL-R BHS. A total of 53(1.3%) BHS were identified among 3,958 in the SENTRY AntimicrobialSurveillance Program (2003-2006) that were TEL-R (MIC, ≧2 μg/ml). 43strains (36 group A, 1 group C, 6 group G) were available for testing,from 20 hospitals in Europe (31 strains), North America (11) and LatinAmerica (1). Susceptibility (S) testing used CLSI broth microdilutionmethods and 3 strains were erythromycin (ERY)-R, CLN (CC)-S requiringD-test. Nine comparison agents were tested (4 in Table).

MIC distributions for CEM-101 as MLSB-macrolide comparisons agents:Occurrences at MIC (μg/ml): Antimicrobial ≦0.015 0.03 0.06 0.12 0.25 0.51 2 4 >4 CEM-101 4 0 1 18 10 6 4 0 0 0 TEL 0 0 0 0 0 0 0 8 16 19Erythro- 0 0 0 0 0 0 0 0 — 43 mycin CLN — — — — 2 1 0 0 — 40 Q/D — — — —37 6 0 0 — 0CEM-101 remained active against all TEL-R (MIC, >2 μg/ml) BHS with allMICs at ≦1 μg/ml (MIC50, 0.12 μg/ml). Highest occurrence of TEL-Rstrains was in Europe (greatest in Italy). CEM-101 warrants furtherdevelopment for infections caused by BHS.

The potency of CEM-101 against each BHS serogroup was the same with anoverall MIC50 and MIC90 of 0.12 and 0.5 μg/ml, respectively. CEM-101activity was 32-fold (MIC50 comparisons) greater than TEL. All strainswere ERY-resistant, but quinupristin/dalfopristin (Q/D) was 100% S.Three CC-S strains (S. pyogenes) were D-test (+) and 2 had (+) inductionof CEM-101. The susceptibility rates for other comparators were:penicillin, tetracycline, ceftriaxone, amoxicillin/clavulanate, andlevofloxacin (100.0%); and tetracycline (46.8%).

Example.

Susceptibility testing in broth: Studies with additional strains(including CA-MRSA): MICs at pH 7.4

Strains MSSA/MRSA CEM-101 AZI CLR TEL ATCC 25923 MSSA 0.125 0.5 0.5 0.5NRS 192 (US) CA-MRSA 0.25 1 0.5 0.25 N4090440 (BE) CA-MRSA 0.06 0.5-10.5 0.5 N7112046 (BE; MRSA 0.06 2 1 0.25 Animal MRSA) STA 44 (Asia)CA-MRSA 0.5 256 128 8 CHU (Asia) CA-MRSA 2 256 256 32 STA 268 (Asia)CA-MRSA 0.5-1 128 128 16 MEH (Asia) CA-MRSA 0.125 1 0.5 0.25

In vivo: Intraperitoneal (IP) - Mouse Infection Models (ED₅₀) MAC-SMAC-R S. pneumoniae S. pyogenes Compound ATCC 49619 (MIC) 3029 (MIC)CEM-101 2.5 mg/kg (<=0.125) 2.5 mg/kg (<=0.125) AZI 15 mg/kg(<=0.125) >150 mg/kg (>64) TEL 1 mg/kg (<=0.125) >150 mg/kg (16)

Example.

Antimicrobial activity was assessed in a murine systemic infection modelagainst several bacterial strains including resistant isolates such asMRSA, MRSA 300, mefR S. pneumoniae, an erythromycin resistant S.pyogenes, and a serotype 19A S. pneumoniae isolate.

Example.

Efficacy was evaluated in several infection models. CD-1 female micewere infected IP; CEM-101 or comparators were administered as a singleoral dose 1 hr post infection. PD50s were determined 24 hr postinfection. CEM-101 was further evaluated in a subcutaneous abscess mousemodel against S. pneumoniae. CD-1 female mice were infected via SCinjection of bacteria mixed with cyclodextran beads. Two hr postinfection, mice received a single oral dose of CEM 101 or controlagents. At 48 hr post dose, mice were euthanized, abscesses asepticallyremoved and bacteria enumerated. CFU per abscess was determined andcompared to the untreated control. Further evaluation of CEM-101 wasperformed in cyclophosphamide induced neutropenic mice. At 1.5 hr postthigh infection with S. pneumoniae, mice were orally dosed with CEM-101or control drugs. 24 hr post treatment, the thighs were processed andCFU/gram of thigh determined.

Mouse Systemic Infection Model (mg/kg) CEM-101 TEL CLR S. aureus16.3  >30    22.7 (11.2-21.3) (11.3-34.1) MRSA 7.5 ND  5.0 (6.0-8.9) S.pneumoniae 6.0 19.9  32.1 (macrolide susceptible)  (2.0-10.0) (9.6-30.2)(12.3-52.0) S. pneumoniae 23.2  10.6  >30   (mef R) (15.6-30.7)(2.6-18.6) S. pyogenes 9.4 7.8 24.8 (macrolide susceptible)  (7.3-11.5)(5.7-9.8)  (18.1-30.4) S. pyogenes 5.1 4.4 22.8 (erythromycin R)(4.2-6.1) (3.8-4.9)  (14.6-30.9) ND = not determined

In the abscess, a 10 mg/Kg QD dose of CEM-101 demonstrated a 4.2 log_(e)decrease while CLR (CL) only achieved a 1.5 log₁₀ reduction fromuntreated mice. CEM-101 in the thigh required 8.0 mg/Kg to achieve a 3log₁₀ reduction from the untreated mice. TEL and CL required 15.5 and13.5 mg/Kg to achieve the same log₁₀ CFU reductions.

Example.

Media. Trypicase Soy Agar (TSA) plates—BBL, Franklin Lakes, N.J.;TiypicaseSoy Agar with 5% sheep blood (TSA-II)-BBL, Franklin Lakes,N.J.; Brain Heart Infusion (BHI) Broth—BBL, Franklin Lakes, N.J.; TypeIII Hog Gastric Mucin-Sigma Aldrich, St Louis Mo.; CyclodextranBeads—Sigma-Aldrich, St. Louis, Mo.; Cyclophosphamide Sigma-Aldrich, St.Louis, Mo.

Example.

Experimental Design. CD-1 Female mice (weighing 18 to 22 grams) fromCharles River Laboratories (Wilmington, Mass.) were acclimated for 5days prior to start of studies. All studies were performed underapproved IACUC protocols and conform to OLAW standards. Animals had freeaccess to food and water throughout the study as well as providedenrichment.

Mouse Systemic Infection Studies. Eight bacterial isolates wereevaluated in this model. For each strain, an overnight culture wasutilized. The bacteria were re-suspended in media and diluted either inBHI, 5% or 8% hog gastric mucin to a concentration that would result in0% survival in mice by 48 hours post infection as determined by initialvirulence studies. Bacterial counts were performed to determine inoculumsize. Mice received treatment via oral gavage 1 hour post infection. Attermination of study, percent survival was calculated and the doseeffecting 50% survival, the protective dose 50% (PD50), was reportedalong with 95% confidence intervals as calculated by Probit analysisusing GraphPad Prism version 4.03 (GraphPad Software).

Example.

Mouse Subcutaneous Abscess. Bacteria were prepared from an overnightplate culture by re-suspending in saline and adjusting the suspension toa 0.1 OD at 625 nm of a 1:10 dilution. The adjusted bacterial suspensionwas mixed 1:2 with cyclodextran beads prepared as per packageinstructions. The right flanks of the mice were shaven and injectedsubcutaneously with 0.2 ml of the bacterial inoculum. Two hours postinfection mice were treated via oral gavage with either test article orcontrol drug. 48 hours post infection, mice were euthanized, abscessesaseptically removed, homogenized, serially diluted and plated onbacterial growth agar. After overnight incubation, colonies were countedand CFUs/gram of abscess were determined.

Example.

Neutropenic Mouse Thigh Infection. Mice were rendered neutropenic withIP injections of cyclophosamide at day −4 and day −1 of 150 mg/Kg and100 mg/Kg, respectively. On day 0 mice were infected with approximately5×10⁵ CFU/ml of bacteria in a 0.1 ml volume into the right thigh. At 1.5hours post infection mice received treatment via oral gavage. One groupof infected mice were euthanized and thigh processed for bacterialtiters to serve as T=0 controls. Twenty-four hours post treatment, theremaining mice were euthanized, thighs aseptically removed, weighed,homogenized, serially diluted and plated on bacterial growth media. CFUsper gram of thigh were calculated after overnight incubation ofbacterial plates. The amount of test article required to achieve 1, 2,and 3 log_(e) reductions from 24 hour control thighs were calculated.Additional studies were performed that fractionated the single treatmentdose (Q24) into two (Q12), three (Q8) and four (Q6) equivalent doses todetermine the pharmacodynamic nature of this compound. Further analysisincludes static dose, EC50, 1 log kill and maximal effect (Emax).

Example.

Pharmacokinetics. Mouse Pharmacokinetics of CEM-101

Dose Tmax Cmax AUC0-24, Half-life (mg/kg) Route (h) (ng/ml) (ng*hr/mL)(h) 2.5 PO 1 359.15 1172.63 2.85 10.0 PO 0.5 1436.60 4757.93 2.85

It is appreciated herein that the subcutaneous abscess model and theneutropenic thigh model in mice described herein may be more relevant tothe understanding and/or predictability of efficacy of the compoundsdescribed herein in treating human infection in vivo than the mouseprotection model, which may be a better model for the understandingand/or predictability of efficacy of the compounds described herein fortreating blood-borne infections, such as bacteremia. For example,relative to CLR and TEL, if a triazole-containing compound, such asCEM-101, has a lower C_(max) in the mouse at the same doses, the MICsare reflected accurately in the mouse protection tests for thesusceptible strains. It is observed that CEM-101 is very active when thedose is adjusted for C_(max) in the mouse. Against resistant strains,only CEM-101 is effective even in the mouse protection tests. Incontrast, in more relevant models of human infection such as thesubcutaneous abscess model or the neutropenic thigh model, CEM-101performs extraordinarily well and reflects its in vitro potency.

Example.

The ability of CEM101 to reduce the microbial load in abscess infectionmodel was also assessed against S. pneumoniae isolates.

Example.

Mouse Systemic Infections.

CEM-101 CLR PD50 PD50 PD50 MIC (mg/Kg; 95% MIC (mg/Kg; 95% MIC (mg/Kg;95% (μg/ml) CI)) (μg/ml) CI)) (μg/ml) CI)) S. aureus 0.12 16.3(11.2-21.3) 0.06 >30 >16 22.7 (11.3-34.1) MRSA 0.12 7.5 (6.0-8.9) 0.5ND >16 5.0 MRSA 300 (CA) 0.12 9.5 (5.0-13.9) 0.25 9.2 (6.3-12.1) >1619.5 (8.2-30.5) S. pneumoniae <0.03 6.0 (2.0-10.0) <0.06 19.9(9.6-30.2) >16 32.1 (12.3-52.0) (macrolide susceptible) S. pneumoniae<0.03 23.2 (15.6-30.7) 0.25 10.6 (2.6-18.6) 0.5 >30 (mefR) S. pneumoniaeserotype 0.25 6.6 (4.4-8.9) 0.5 5.7 (4.8-6.7) >16 5.03 (4.8-5.3) 19A S.pyogenes 0.015 9.4 (7.3-11.5) 0.015 7.8 (5.7-9.8) 0.015 24.8 (18.1-30.4)(macrolide susceptible) S. pyogenes 1.0 5.1 (4.2-6.1) 1.0 4.4 (3.8-4.9)1.0 22.8 (14.6-30.9) (erythromycin R) ND = not determined

Example.

Mouse Subcutaneous Abscess ˜S. pyogenes ATCC 8668 (Average Log₁₀CFU/gram of Abscess). Abscess processing 48 hours post infection.

Dose (mg/Kg) 48 hr. Control — 8.22 CEM-101 10 5.35 20 2.53 CLR 10 7.6320 7.82 TEL 10 7.06 20 6.10

Example.

Neutropenic Thigh Model. Mouse Neutropenic Thigh S. pneumoniae 1629(Dose (mg/Kg) QD, PO at 2 h post infection; Reduction from 24 hourcontrols).

Max. change in log10CFUs Dose 1 log10 2 log10 3 log10 from 24 CompoundRoute Reduction Reduction Reduction hr. controls CEM-101 PO 6.0 7.0 8.0−5.81 TEL PO 11.0 13.2 15.5 −6.82 CLR PO 4.5 9.0 13.5 −5.75

In the equivalent Mouse Subcutaneous Abscess ˜S. pneumoniae 1629 (10mg/Kg PO @ 2 hours post infection) CEM-101 and CLR average log₁₀CFU/gram of absess remained constant at about 3 and 5.5, respectivelyfrom 24 hours to 48 hours; TEL increased from about 3 to about 4.5.

Example.

Mouse Neutropenic Thigh S. pneumoniae 6303 (Dose (mg/Kg) QD, PO;Reduction from 24 hour controls)

Max. change in log10CFUs Dose 1 log10 2 log10 3 log10 from 24 CompoundRoute Reduction Reduction Reduction hr. controls CEM-101 PO 1.2 3.0 5.0−6.17 TEL PO 4.75 6.5 8.75 −5.27 CLR PO 5.5 9.2 14.0 −5.20

Example.

Mouse Neutropenic thigh Model Fractionated dosing studies: S. pneumoniae6303 vs. CEM-101 PO

Q24 Q12 Q8 Q6 Static dose (mg/Kg) 8.2 19.2 12.1 20.5 EC50(mg/Kg) 5.714.5 9.0 16.7 1 log kill (mg/Kg) 10.8 24.0 23.0 23.0 Emax(log10CFU/thigh) 6.38 4.95 5.01 5.83

Example.

Mouse Lung Infection. The ability of CEM 101 to reduce the microbialload in lung infection model was also assessed against S. pneumoniaeisolates. Bacteria were prepared from an overnight plate culture byre-suspending in saline and adjusting the suspension to a 0.1 OD at 625nm of a 1:10 dilution. Mice, under light anesthesia, were inoculatedwith 50 μl of the S. pneumoniae1629 bacterial inoculum via intranasalinhalation. Mice received treatment via oral gavage 5, 24, and 36 hourspost infection. 48 hours post end of treatment, mice were euthanized,lungs aseptically removed, homogenized, serially diluted and plated onbacterial growth agar. After overnight incubation, colonies were countedand CFUs/gram of lung were determined Average log_(in) CFU/gram of lungwas increased more in the CLR treated mice, from 3 to greater than 7,compared to CEM-101 treated mice, from 5 to about 5.5, from 24 hours to48 hours.

Example.

A worldwide sample of organisms included S. pneumoniae (SPN; 168, 59.3%erythromycin [ERY]-R and 18 multidrug-resistant [MDR]-19A strains), M.catarrhalis (MCAT; 21, 11β-lactamase[+]), H. influenzae (HI; 100,48β-lactamase[+]), H. parainfluenzae and H. haemolyticus (12) andLegionella pneumophila (LPN; 30). All S tests were by reference CLSImethods (M7-A7, M100-S18) and breakpoints per CLSI (2008) for comparisonagents such as AZI (AZ), CLR (CLR), ERY, TEL (TEL), CLN (CC), Synercid®(SYN), levofloxacin (LEV), linezolid, and rifampin (RIF). SPN were verysensitive to CEM (MIC₉₀, 0.25 μg/ml; highest MIC at 0.5 μg/ml) and CEMwas 2- and 8-fold more potent than TEL and CC, respectively.

Eighteen serogroup 19A strains exhibited high levels ofnonsusceptibility to: macrolides (100.0%), CLN (83.3%), penicillin(83.3%), amox/clav (88.9%), ceftriaxone (33.3%), tetracyclines (83.3%and TMP/SMX (100.0%). Few therapeutic options remain with only TEL(MIC₉₀, 1 μg/ml; 100.0% susceptible), Q/D (MIC₉₀, 1 μg/ml; 100.0%susceptible) and fluoroquinolones (MIC₉₀, 1 μg/ml; 100.0% susceptible)having usable potencies (Table 3). CEM-101 showed a potency two-foldgreater than TEL.MDR-19A replacement strains were also CEM-S (MIC₉₀, 0.5μg/ml), compared to TEL (MIC₉₀, 1 μg/ml), ERY (MIC₉₀, >32 μg/ml), AZI(MIC₉₀, >16 μg/ml), CLR (MIC₉₀, >32 μg/ml), and CLN (MIC₉₀, >16 μg/ml).

LPN were most S to CEM with all MIC values at ≦0.015 μg/ml (TEL MIC90,0.03 μg/ml). Haemophilus RTI pathogens were less CEM-S (MIC₉₀, CEM/TEL):HI (2/4 μg/ml) and others (2/4 μg/ml) with no variations for β-lactamase(+) strains. MCAT CEM-101 MICs were all at ≦0.5 μg/ml, equal to TEL.

S. pneumoniae were very susceptible to CEM-101 with a MIC₉₀ of only 0.25μg/ml. This documented potency (Table 1) was two-fold greater than TELand eight-fold superior to linezolid (MIC₉₀, 2 μg/ml). β-haemolyticstreptococci were also susceptible to CEM-101 (MIC₉₀, 0.03 μg/ml) withthis new agent showing a four-fold advantage (MIC90, 0.12 μg/ml; 100.0%susceptibility) over TEL. Five groups of β-haemolytic strains weretested and all strains showed a monomodal MIC (0.015 μg/ml) distributionand the highest CEM-101 MIC was only 0.12 μg/ml (Table 1).

CEM-101, like TEL, was active against all macrolide- and CLN-resistantviridans group streptococci (five species groups; 51 strains), but allCEM-101 MIC values were at ≦0.12 μg/ml, four-fold more potent than TELand 64-fold more active than erythromycin.

All CEM-101 MIC results for Haemophilus spp, had a narrow range of only0.5-4 μg/ml (exception two strains of 0.12 μg/ml that did not exhibit anefflux pump). The overall MIC₉₀ for strains in this genus was 2 μg/ml,equal to AZI and two-fold more active than TEL. The various species (H.influenzae, H. parainfluenzae) and β-lactamase production did notsignificantly alter CEM-101 activity (MIC₉₀, 2 μg/ml; Table 1).

Among the MLSB agents, the rank order of potency (MIC₉₀ in μg/ml)against M. catarrhalis was: AZI (0.06)>CEM-101=CLR(0.12)>erythromycin=TEL (0.25)>Q/D (0.5)>CLN (2; see Table 1). Theβ-lactamase activity had no significant effect on the CEM-101 MIC₉₀values (0.12 μg/ml).

CEM-101 (MIC₉₀, ≦0.015 μg/ml) was the most active agent tested againstLegionella spp. (Table 1), superior to other macrolides, levofloxacinand rifampin. Note that the charcoal content of the test media caninterfere with the reference MIC testing of this species.

CEM MIC TEL MIC (μg/ml) (μg/ml) Organism (no.) 50% 90% Range 50% 90%Range SPN (150) 0.015 0.25 ≦0.008-0.5 0.03 0.5 ≦0.008-1   MDR-19A (18)0.25 0.5  0.06-0.5 0.5 1 0.12-1 MCAT (21) 0.12 0.12 ≦0.008-0.5 0.12 0.25≦0.015-0.5   HI (100) 1 2  0.12-4 2 4  0.25-16 Other Haemophilus (12) 22  0.12-2 2 4 0.25-8 LPN (30) ≦0.015 ≦0.015 ≦0.015 0.03a 0.03a   0.03-0.06^(a) 1. RIF results, not TEL.

Screening in vitro studies of the compounds described herein indicates apotency comparable or superior to TEL, ERY, AZI and CLR, as well asactivity against Gram-positive isolates having documented resistances tomacrolides or lincosamides. CEM-101 activity is generally focusedagainst Gram-positive pathogens, but also possesses measurable potenciesversus fastidious Gram-negative species (Haemophilus, Moraxella), someEnterobacteriaceae (Salmonella, Shigella) and pathogens causing varioussexual transmitted diseases (STD). CEM-101 activity was measured byreference Clinical and Laboratory Standards Institute (CLSI) methodswhen testing organisms associated with CARTI (streptococci, Haemophilusspp., Moraxella catarrhalis, Legionella pneumophila), emerging resistantsubsets (serogroup 19A S. pneumoniae) and various patterns ofMLSB-ketolide resistance among the tested streptococci.

Organism collection: All organisms tested were collected from patientsin the USA and European medical centers from 2005 to present. Sources ofrecovered isolates included bloodstream, skin and soft tissue andrespiratory tract infections. Unusual/rare organism species andphenotypes required use of strains isolated prior to 2005 or from othergeographic areas. Organisms tested: Streptococci (319), S. pneumoniae(150 wild type), S. pneumoniae (18 serogroup 19A, USA only),β-haemolytic species (100, five groups), viridans group (51, fivespecies), Haemophilus species (111), H. influenzae (100, 48β-lactamaseproducers), H. parainfluenzae (11), M. catarrhalis (21, 11β-lactamaseproducers), L. pneumophila (30).

Susceptibility testing: Ninety-six well frozen-form assay panels wereproduced by JMI Laboratories and consisted of three media types:cation-adjusted Mueller-Hinton broth, cation adjusted Mueller-Hintonbroth with 2.5-5% lysed horse blood (for testing streptococci) andHaemophilus Test Medium (HTM). CLSI broth microdilution and agardilution methods per M7-A7 [2006] were used. Quality control (QC) rangesand interpretive criteria for comparator compounds were those publishedin CLSI M100-S18 [2008]. Tested QC strains included S. aureus ATCC29213, E. faecalis ATCC 29212, S. pneumoniae ATCC 49619 and H.influenzae ATCC 49247 and 49766. All QC results were within publishedlimits. Agar dilution methods were used for L. pneumophila tested onBCYE agar. Comparison agents were tested by Etest, also on BCYE media. Awide variety of comparison agents were utilized including:amoxicillin/clavulanate (amox/clay), AZI, cefdinir, CLR, CLN,erythromycin, levofloxacin, linezolid, quinupristin/dalfopristin (Q/D),TEL and trimethoprim/sulfamethoxazole (TMP/SMX) all assessed by brothmicrodilution; and ciprofloxacin, tetracycline, ampicillin and rifampinwere additionally tested on agar.

The Table shows excellent CEM-101 potency against streptococci (allMICs, ≦0.5 μg/ml) and moderate activity against Gram-negative CA-RTIpathogens (MICs, ≦0.008-4 μg/ml).

CEM-101 MIC distributions for all tested RTI organisms (398 strains)showing Occurrences at MIC (μg/ml). Organism (no. tested) ≦0.008 0.0150.03 0.06 0.12 0.25 0.5 1 2 4 8 ≧16 S. pneumoniae (150) 62 25 8 9 7 33 60 0 0 0 0 β-haemolytic streptococci (100) 21 65 4 8 2 0 0 0 0 0 0 0Viridans group streptococci (15) 27 11 4 7 2 0 0 0 0 0 0 0 M.catarrhalis (21) 1 1 1 5 12 0 1 0 0 0 0 0 H. influenzae (100) 0 0 0 0 10 5 48 42 4 0 0 Haemophilus, other (12) 0 0 0 0 1 0 0 4 7 0 0 0

CEM-101 exhibited potent activity against streptococci (MIC50, 0.015μg/ml), and various other Gram-positive cocci including strainsresistant to erythromycin and CLN. CEM-101 showed complete activityagainst MDR serogroup 19A pneumococci (16 of 18 MIC values at 0.25 or0.5 μg/ml) and was two-fold more active than TEL and Q/D. CEM-101 alsoinhibited Gram-negative species associated with CA-RTI (H. influenzae[MIC90, 2 μg/ml], Legionella spp. [MIC90, ≦0.015 μg/ml], and M.catarrhalis [MIC90, 0.12 μg/ml]).

Example.

Macrolide resistant isolates (29) (based on CLR MICs determination; 19MLSB, 10 M-phenotype based on erythromycin and CLN resistancedissociation) were selected (for which 6 were TEL-1 and 7 TEL-R based onEUCAST breakpoints [S<=0.25-R>0.5]). MICs were determined by geometricmicrodilution in CAMH broth+2.5% lysed horse blood according to CLSI,using SP ATCC-49619 as a control.

Example.

ATCC-49619 MICs were ≦0.008 mg/L for TEL and CEM-101. Data forML-resistant isolates are shown in the Table. In this Belgian collectionof S. pneumoniae from confirmed CAP resistant to macrolides, CEM-101shows globally lower MICs compared to TEL, especially with respect toTEL-I and TEL-R isolates.

TEL CEM-101 Phenotype* No. range geom. mean MIC₉₀ range geom. mean MIC₉₀TEL-S 16 0.008-0.25  0.021 0.25 0.008-0.063 0.022 0.063 TEL-I 6 0.5-0.50.5 0.5 0.063-0.5  0.223 0.5 TEL-R 7 1-3 1.426 3.0 0.5-1.0 0.906 1.0*MLS_(B) for 7/16 of TEL-S, 5/6 pf TEL-I, and 7/7 of TEL-R isolates(S/I/R are defined based on EUCAST breakpoints (S ≦ 0.25 − R > 0.5)

CEM-101 shows globally lower MICs compared to TEL, especially withrespect to TEL-I and TEL-R isolates tested against this Belgiancollection of S. pneumoniae from confirmed cases of CAP which areresistant to macrolides. As described herein, CEM-101 has the potentialto be useful as an alternative to telithromcyin in areas with high MLresistance and emerging resistance to TEL.

S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniaeATCC 49619 ErmB 303 163 (Mef A) 3773 (Erm B) 5032 AZI ≦0.125 >648 >64 >64 TEL ≦0.125 ≦0.125 ≦0.125 1 0.5 CEM-101 ≦0.125 ≦0.125 ≦0.1250.5 0.5

S. pyogenes S. pyogenes S. pyogenes S. pyogenes H. inflluenzae 1721 18503029 3262 ATCC 49247 AZI >64 >64 >64 >64 2 TEL 64 8 16 32 4 CEM-101 0.5≦0.125 ≦0.125 0.5 2

MIC₅₀ and MIC₉₀ [μg/mL] CEM-101 OP 1055 TEL # Of Strains MIC₅₀ MIC₉₀MIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ S. pneumoniae 10 0.03 0.03 0.03 0.03 0.03 0.03PEN-S, TEL S; Macr-S S. pneumoniae 12 0.5 0.5 4 4 2 8 PEN-R, TEL I/R;Macr-R S. pneumoniae 24 0.03 0.03 0.06 0.06 0.06 0.06 PEN-S, TEL S;Macr-R S. pyogenes 10 0.03 0.03 0.03 0.03 0.03 0.03 TEL S; Macr-S S.pyogenes 10 0.125 0.25 2 2 32 32 TEL R; Macr-R S. pyogenes 30 0.03 0.030.125 0.25 0.125 0.5 TEL S; Macr-R

# Of 2-F-TEL AZI PEN Strains MIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ S.pneumoniae 10 0.03 0.03 0.03 0.03 0.03 0.03 PEN-S, TEL S; Macr-S S.pneumoniae 12 1 8 >32 >32 4 8 PEN-R, TEL I/R; Macr-R S. pneumoniae 240.03 0.06 >32 >32 0.03 0.03 PEN-S, TEL S; Macr-R S. pyogenes 10 0.030.03 0.125 0.125 0.03 0.03 TEL S; Macr-S S. pyogenes 10 32 32 >32 >320.03 0.03 TEL R; Macr-R S. pyogenes 30 0.25 0.5 >32 >32 0.03 0.03 TEL S;Macr-R

Example.

Where on-scale MIC results were available, CEM-101 (MIC90, 0.015 μg/ml)was two- and eight-fold more active than CLR or TEL and AZI,respectively, against streptococci susceptible to erythromycin. Forstreptococci non-susceptible to erythromycin, CEM-101 had an elevatedMIC (MIC90, 0.25 μg/ml); however, CEM-101 was at least two-fold moreactive than either TEL or CLN. When tested against erythromycin- andCLN-non-susceptible streptococci, all but one strain remainedsusceptible to TEL, but all were inhibited by CEM-101 at ≦0.5 μg/ml.

Activity of CEM-101 and selected comparison agents against streptococcihaving various MLS_(B) resistance patterns (three groups, 300 strains*).MLS_(B)-ketolide Anti- resistance pattern microbial MIC (μg/ml) (No.tested)^(a) agent 50% 90% Range ERY-S (164)^(b) CEM-101 ≦0.008  0.015≦0.008-0.03 TEL 0.03 0.03   0.008-0.06 CLR 0.03 0.03 ≦0.008-0.25 AZI0.06 0.12 ≦0.008-0.5  CLN ≦0.12   ≦0.12 ≦0.12-0.5 Q/D 0.5  0.5 ≦0.12-2  Amox/clav ≦0.25   2 ≦0.25->4  Levofloxacin 1   1  0.25->4 Linezolid 1  2 0.25-2 ERY-NS, CLN-and CEM-101 0.03 0.12 ≦0.008-0.25 TEL-S (48)^(c)TEL 0.12 0.5 0.03-1 CLN ≦0.12   0.25  ≦0.12-0.25 Q/D 0.5  1 0.25-2Amox/clav ≦0.25   2 ≦0.25->4  Levofloxacin 1   2  0.25->4 Linezolid 1  2  0.5-2 ERY-and CLN-NS, CEM-101 0.06 0.25 ≦0.008-0.5  TEL-S (88)^(d)TEL 0.12 1 0.03-1 Q/D 0.5  1 0.25-2 Amox/clav 2   >8 ≦0.25->8Levofloxacin 1   1 0.25-2 Linezolid 1   1  0.5-2 ^(a)S = susceptible andNS = non-susceptible e.g. includes intermediate and resistant strains.^(b)Includes: Streptococcus anginosus (9 strains), S. constellatus (8strains), S. intermedius (7 strains), S. mitis (2 strains), S. oralis (3strains), S. pneumoniae (61 strains), Group A (29 strains), Group B (17strains), Group C (11 strains), Group F (6 strains) and Group GStreptococcus (11 strains). ^(c)Includes: Streptococcus anginosus (2strains), S. constellatus (2 strains), S. intermedius (3 strains), S.mitis (7 strains), S. oralis (7 strains), S. pneumoniae (14 strains),Group A (1 strain), Group B (7 strains), and Group G Streptococcus (5strains), ^(d)Includes: Streptococcus constellatus (1 strain), S.pneumoniae (75 strains), Group B (7 strains), Group C (2 strains), andGroup F Streptococcus (3 strains). One strain (group C streptococcus)was NS to ER, CLN and TEL, but had a CEM-101 MIC at 0.06 μg/ml. This isan important emerging pattern, especially in EuropeOne strain (group C streptococcus) was NS to ER, CLN and TEL, but had aCEM-101 MIC at 0.06 μg/ml. This is an important emerging pattern,especially in Europe

Example.

Macrolide-Susceptible And Macrolide-Resistant Streptococci. The activityof CEM-101 compared to those of erythromycin, AZI, CLR, TEL, CLN,penicillin G, amoxicillin/clavulanate, levofloxacin, and moxifloxacinagainst a range of pneumococci was tested. The potency of CEM-101,erythromycin, AZI, CLR, TEL, CLN, penicillin G, amoxicillin/clavulanate,levofloxacin and moxifloxacin against 124 S. pyogenes strains was alsotested.

Pneumococcal MIC50 and MIC90 values (μg/ml) Macrolide Macrolidesusceptible (50) resistant (171) Drug Range MIC50 MIC90 Range MIC50MIC90 CEM- 0.002-0.015 0.03 0.25 0.004-1 0.06 0.25 101 ERY 0.03-0.250.06 0.125    1->64 >64 >64 AZI 0.06-0.25 0.125 0.125    1->64 >64 >64CLR 0.015-0.06  0.03 0.06  0.25->64 32 >64 TEL 0.015-0.03  0.03 0.03 0.03-2 0.125 0.5 CLN 0.015-0.06  0.03 0.06  0.03->64 0.125 >64 Amox/0.015-8    0.5 2 0.015-16 1 8 clav Pen G 0.015-8    1 2 0.008->16 1 4Levo  1-32 1 16  0.06-32 1 2 Moxi 0.125-8    0.25 4 0.125-4 0.25 0.5

MICs for group A streptococci Macrolide Macrolide susceptible (26)resistant (98) Drug Range MIC50 MIC90 Range MIC50 MIC90 CEM-1010.008-0.03  0.015 0.3 0.015-1   0.06 0.5 ERY 0.03-0.25 0.06 0.125  2->64 16 >64 AZI 0.06-0.25 0.125 0.25   0.5->64 8 >64 CLR 0.015-0.06 0.03 0.06  0.25->64 4 >64 TEL 0.03-0.06 0.06 0.06 0.03-16  0.25 8 CLN 0.03-0.125 0.06 0.06  0.03->64 0.125 >64 Amox/clav 0.015-0.03  0.030.03 <0.015-0.125 0.03 0.03 Pen G <0.008-0.125  0.015 0.015 <0.008-0.1250.015 0.015 Levo 0.5-1   0.5 0.5 0.5-2  0.5 1 Moxi 0.125-0.25  0.25 0.250.125-0.5  0.25 0.25

All group A streptococcal strains were penicillin G susceptible. Againstmacrolide susceptible strains, CEM-101 MICs were 0.008-0.03 μg/ml andthose against macrolide resistant strains (all phenotypes) MICs were0.008-0.5 μg/ml. TEL MICs were up to four fold higher than those ofCEM-101. Importantly, 11/13 erm(B) strains were TEL resistant with MICsof 4 and 8 μg/ml (6) while all had low CEM-101 MICs, similar to those ofother resistance phenotypes (range 0.016-0.5 μg/ml).

Example.

Bacteria and Antimicrobials. MICs were determined for 221 clinicalpneumococcal strains, including 50 macrolide-susceptible and 171macrolide-resistant organisms. Macrolide-resistant strains all haddefined genotypes and comprised strains with erm(B) (54 strains), mef(A)(51 strains), erm(B)+mef(A) (31 strains), erm(A) (4 strains) andmutations in L4 ribosomal protein (27 strains), and 23S rRNA (4strains). These 221 strains also included 27 quinolone non-susceptiblephenotypes with defined quinolone resistance determinant regions (QRDRs)(levofloxacin MICs 4-32 μg/ml) and the entire spectrum of penicillin Gresistance phenotypes using the latest Clinical Laboratory StandardsInstitute (CLSI) oral penicillin V susceptibility classification (4).The 124 group A streptococci tested by MIC included 26macrolide-susceptible and 98 macrolide-resistant strains. The latterincluded 19 strains with erm(B), 38 mef(A), 40 erm(A) and 1 strain withan L4 mutation. Because strains of both species were chosen for theirmacrolide resistance phenotype, only susceptible strains wereconsistently recent (2003-2008) isolates; some resistant strains wereisolated up to five years earlier (1998). Streptococcus pneumoniae ATCC49619 was included as the quality control strain for each species ineach run.

For resistance selection testing, one each of the following pneumococcalresistance phenotypes was tested: macrolide-susceptible, erm(B)positive, mef(A) positive, erm(B)+mef(A) positive, erm(A) positive andwith mutation in ribosomal proteins (L4, L22) and 23S rRNA. Five strainsof group A streptococci were tested with one each macrolide-susceptible,erm(B) positive, mef(A) positive, erm(A) positive and with L4 mutation.

MICs were done by the agar dilution technique which. Mueller-Hinton agar(BD Diagnostics, Sparks, Md.)+5% sheep blood agar was used, with 10⁴CFU/spot and overnight incubation at 35° C. in ambient air. The usualquality control strains were included in each run. For resistanceselection, CLSI macrodilution was used for MIC testing.

All macrolide-resistant parental strains were tested for the presence ofthe erm(B), erm(A), mef(E) and mef(A) genes by PCR amplification. Thepresence of mutations in the L4 and L22 ribosomal proteins and 235 rRNA(II and V domain) were examined in all parental isolates and in CEM-101resistant clones (CEM-101 MIC>1 μg/ml.) using conventional primers andconditions. The nucleotide sequences were obtained by direct sequencingwith a CEQ8000 genetic analysis system (Beckman. Coulter, Fullerton,Calif.).

Serial passages were performed daily from each strain in sub-inhibitoryconcentrations of all antimicrobials. In all cases, broth medium was 1ml per tube of cation-adjusted Mueller-Hinton broth (BD Diagnostics,Sparks, Md.)+5% lysed horse blood. For each subsequent daily passage, aninoculum (10 μl) was taken from the tube one to two dilutions below theMIC that matched the turbidity of a growth control tube. This inoculumwas used to determine the next MIC. Daily passages were performed untila significant increase in MIC (≧8 times) was obtained. A minimum of 14passages was performed unless MICs≧32 μg/ml were obtained. The maximalnumber of passages was 50. Stability of the acquired resistance wasdetermined by MIC determinations after 10 daily passages of the mutantson blood agar without antibiotics. MICs of each resistant pneumococcalclone to each compound were determined by macrodilution MIC. Identity ofthe obtained mutants and their respective parents was confirmed bypulsed-field gel electrophoresis (PFGE) at the end of the study. PFGE ofSmaI digested DNA was performed using a CHEF DR III apparatus (Bio-Rad,Hercules, Calif.) with the following run parameters: switch time of 5 to20 s and a run time of 16 h.

The frequency of spontaneous single step mutations was determined byspreading suspensions (approximately 10¹⁰ CFU/ml) on Mueller Hinton Agar(BD Diagnostics, Sparks, Md.) with 5% sheep blood at 2, 4 and 8×MIC.After incubation at 35° C. in 5% CO₂ for 48 h, the resistance frequencywas calculated as the number of colonies with MICs increased at least 4×parental MIC per inoculum. Single step studies were not performed withAZI, CLR, CLN and TEL for strains with MICs≧4 μg/ml. Results ofpneumococcal MIC testing are presented in the following Tables.

MICs (μg/ml) of drugs against pneumococcal strains Drug MIC range MIC₅₀MIC₉₀ Penicillin G (221^(a))  0.008->16 1 4 Penicillin S (53)  0.008-0.06 .03 .06 Penicillin I (63) 0.125-1 .5 1 Penicillin R (105)    2->16 4 8 Macrolide S (50) 0.015-8 1 2 erm(B) (54)  0.03-16 1 4mef(A) (51) 0.008-4 0.125 4 erm(A) (4)    0.03-0.03 — — erm(B) + mef(A)(31)  0.03-8 2 4 L4 (27)     1->16 4 16 23S rRNA (4)   0.015-0.5 — —Quinolone S (195)  0.008->16 1 4 Quinolone R (27) 0.015-8 0.25 4 CEM-1010.002-1 0.03 0.25 Penicillin S   0.002-0.25 0.03 0.125 Penicillin I  0.002-0.25 0.03 0.25 Penicillin R 0.004-1 0.06 0.25 Macrolide S   0.002-0.015 0.008 0.015 erm(B) 0.004-1 0.03 0.5 mef(A)   0.008-0.250.03 0.125 erm(A)    0.008-0.015 — — erm (B) + mef(A) 0.015-1 0.125 0.25L4    0.03-0.125 0.06 0.125 23S rRNA   0.002-0.03 — — Quinolone S0.002-1 0.03 0.25 Quinolone R   0.004-−.25 0.008 0.06 Erythromycin  0.03->64 64 >64 Penicillin S   0.03->64 4 >64 Penicillin I  0.03->64 >64 >64 Penicillin R   0.03->64 >64 >64 Macrolide S   0.03-0.25 0.06 0.125 erm(B)    16->64 >64 >64 mef(A)     1->64 4 32erm(A)    2-4 — — erm(B) + mef(A)     4->64 >64 >64 L4     4->64 >64 >6423S rRNA     8->64 — — Quinolone S   0.03->64 >64 >64 Quinolone R  0.03->64 0.06 >64 AZI   0.06->64 16 >64 Penicillin S   0.06->64 4 >64Penicillin I   0.06->64 >64 >64 Penicillin R   0.06->64 >64 >64Macrolide S    0.06-0.25 0.125 .0125 erm(B)    >64->64 >64 >64 mef(A)    1->64 4 8 erm(A)    2-8 — — erm (B) + mef(A)     2->64 >64 >64 L4    2->64 >64 >64 23S rRNA    32->64 — — Quinolone S   0.06->64 >64 >64Quinolone R   0.06->64 0.125 >64 CLR  0.125->64 8 >64 Penicillin S 0.015->64 1 >64 Penicillin I   0.03->64 16 >64 Penicillin R  0.015->6416 >64 Macrolide S   0.015-0.06 0.03 0.06 erm(B)     4->64 >64 >64mef(A)   0.5-32 2 4 erm(A)   0.25-0.5 — — erm (B) + mef(A)    1->64 >64 >64 L4    1-32 16 32 23S rRNA    8-16 — — Quinolone S 0.015->64 16 >64 Quinolone R  0.015->64 0.03 >64 TEL 0.015-2 0.06 0.5Penicillin S 0.015-1 0.06 0.25 Penicillin I 0.015-1 0.06 0.5 PenicillinR 0.015-2 0.125 0.5 Macrolide S   0.015-0.03 0.03 0.03 erm (B)  0.03-20.06 1 mef(A)   0.03-0.5 0.125 0.25 erm (A)    0.03-0.06 — — erm (B) +mef(A)  0.03-2 0.5 1 L4    0.06-0.25 0.125 0.25 23S rRNA    0.03-0.06 —— Quinolone S 0.015-2 0.125 0.5 Quinolone R 0.015-1 0.03 0.125 CLN 0.015->64 0.06 >64 Penicillin S   0.03->64 0.06 >64 Penicillin I  0.03->64 0.125 >64 Penicillin R  0.015->64 0.06 >64 Macrolide S  0.015-0.06 0.03 0.06 erm (B)   0.06->64 >64 >64 mef(A)    0.03-0.1250.06 0.06 erm (A)   0.125-0.25 — — erm (B) + mef(A)   0.03->64 0.06 >64L4    0.03-0.125 0.06 0.125 23S rRNA  0.03-1 — — Quinolone S  0.015->640.06 >64 Quinolone R  0.03-64 0.03 64 Amoxicillin/  0.015-16 0.05 8Clavulanate Penicillin S    0.015-0.125 0.03 0.06 Penicillin I  0.03-20.5 1 Penicillin R  0.125-16 2 8 Macrolide S 0.015-8 0.5 2 erm(B)0.015-8 0.5 8 mef(A) 0.015-8 0.125 2 erm (A)    0.03-0.03 — — erm (B) +mef(A)  0.03-16 2 8 L4 0.125-8 4 8 23S rRNA    0.03-0.06 — — Quinolone S 0.015-16 1 8 Quinolone R 0.015-4 0.5 2 Levofloxacin  0.06-32 1 8Penicillin S  0.06-32 1 16 Penicillin I    1-32 1 2 Penicillin R  0.5-16 1 2 Macrolide S    1-32 1 16 erm (B)   0.5-32 1 2 mef(A)  0.5-81 2 erm (A)    1-1 — — erm (B) + mef(A)    1-16 1 16 L4   0.5-16 1 2 23SrRNA  0.06-1 — — Quinolone S  0.06-2 1 2 Quinolone R    4-32 16 16Moxifloxacin 0.125-8 0.25 2 Penicillin S 0.125-8 0.5 4 Penicillin I0.125-4 0.25 0.5 Penicillin R 0.125-4 0.25 0.5 Macrolide S 0.125-8 0.254 erm (B) 0.125 0.25 0.5 mef(A) 0.125-4 0.25 0.5 erm (A)   0.25-0.5 — —erm (B) + mef(A) 0.125-2 0.25 0.5 L4 0.125-4 0.25 0.5 23S rRNA  0.25-0.5 — — Quinolone S 0.125-1 0.25 0.5 Quinolone R  0.5-8 4 4^(a)No. strains tested.

MIC50 and MIC90 values (μg/ml) of pneumococcal strains with definedmacrolide-resistant mechanism

erm(B) + mef(A) L4 erm(B) (54) mef(A) (51) (31) mutations (27) DrugMIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ MIC₅₀ MIC₉₀ CEM-101 0.03 0.5 0.030.125 0.125 0.25 0.06 0.125 Erythromycin >64 >64 4 32 >64 >64 >64 >64AZI >64 >64 4 8 >64 >64 >64 >64 CLR >64 >64 2 4 >64 >64 16 32 TEL 0.06 10.125 0.25 0.5 1 0.125 0.25 CLN >64 >64 0.06 0.06 0.06 >64 0.06 0.125Amoxicillin 0.5 8 0.125 2 2 8 4 8 Clavulanate Levofloxacin 1 2 1 2 1 161 2 Moxifloxacin 0.25 0.5 0.25 0.5 0.25 0.5 0.25 0.5 Penicillin G 1 40.125 4 2 4 4 16

CEM-101 had an MIC range against macrolide-susceptible pneumococci of0.002-0.015 μg/ml and a range against macrolide-resistant pneumococci(all phenotypes) of 0.004-1 μg/ml. Only 3 strains with erm(B) [with andwithout mef(A)] had CEM-101 MICs of 1.0 μg/ml and 218/221 strains hadCEM-101 MICs of ≦0.5 μg/ml. By contrast, corresponding TEL MIC rangeswere 0.015-0.03 μg/ml for macrolide-susceptible and 0.015-2 μg/ml formacrolide-resistant strains. CEM-101 MICs were up to four-fold lowerthan those of TEL against macrolide-susceptible and -resistant strains.All group A streptococcal strains were penicillin G-susceptible. MICsare presented in the following Tables,

MICs (μg/ml) of drugs against group A streptococci Drug MIC Range MIC 50MIC 90 CEM - 101 (124^(a)) 0.008-1   0.06 0.5 Macrolide S (26)0.008-0.03 0.015 0.03 Erm(B) (19) 0.03-1   0.5 1 Mef(A) (38)  0.06-0.250.125 0.25 Erm(A) (40) 0.016-0.5  0.03 0.125 L4 (1) 0.06 — —Erythromycin  0.03->64 16 >64 Macrolide S  0.03-0.25 0.06 0.125 Erm(B) >64->64 >64 >64 Mef(A)  8-32 16 32 Erm(A)   2->64 4 >64 L4 2   — — AZI 0.06->64 8 >64 Macrolide S  0.06-0.25 0.125 0.25 Erm(B) >64->64 >64 >64 Mef(A)  0.5-16 8 8 Erm(A)   2->64 16 >64 L4 2   — — CLR0.015->64  4 >64 Macrolide S 0.015-0.06 0.03 0.06 Erm(B)  32->64 >64 >64Mef(A) 0.5-8  4 8 Erm(A)  0.25->64 2 >64 L4 1   — — TEL 0.03-16  0.125 8Macrolide S  0.03-0.06 0.06 0.06 Erm(B) 0.03-16  8 16 Mef(A) 0.125-1  0.5 1 Erm(A)  0.03-0.25 0.06 0.125 L4 0.06 — — Amoxicillin/Clavulanate<0.015-0.125 0.03 0.03 Macrolide S 0.015-0.03 0.03 0.03 Erm(B)<0.015-0.125 <0.015 0.03 Mef(A) <0.015-0.125 0.015 0.06 Erm(A)<0.015-0.03  0.03 0.03 L4 0.03 — — Levofloxacin 0.5-2  0.5 1 Macrolide S0.5-1  0.5 0.5 Erm(B) 0.5-1  0.5 1 Mef(A) 0.5-2  0.5 1 Erm(A) 0.5-2  0.51 L4 0.5  — — Moxifloxacin 0.0125-0.5  0.25 0.25 Macrolide S 0.125-0.250.25 0.25 Erm(B) 0.125-0.25 0.25 0.25 Mef(A) 0.25-0.5 0.25 0.25 Erm(A)0.125-0.5  0.25 0.25 L4 0.25 — — Pen G <0.008-0.125 0.015 0.015Macrolide S  0.008-0.015 0.015 0.015 Erm(B) <0.008-0.125 0.015 0.015Mef(A) <0.008-0.125 0.015 0.03 Erm(A) <0.008-0.015 0.015 0.015 L4  0.015— — CLN  0.03->64 0.125 >64 Macrolide S  0.03-0.125 0.06 0.06 Erm(B) 0.06->64 >64 >64 Mef(A)  0.03-0.125 0.06 0.125 Erm(A) 0.06-0.5 0.1250.25 L4 0.06 — — ^(a)Number of strains tested

MIC50 and MIC90 values (μg/ml) of group A streptococcal strains withdefined macrolide-resistant mechanisms erm(B) mef(A) erm(A) (19) (38)(40) Drug MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 CEM-101 0.5 1 0.125 0.250.03 0.125 Erythromycin >64 >64 16 32 4 >64 AZI >64 >64 8 8 16 >64CLR >64 >64 4 8 2 >64 TEL 8 16 0.5 1 0.06 0.125 CLN >64 >64 0.06 0.1250.125 0.25 Amoxicillin/ <0.015 0.03 0.015 0.06 0.03 0.03 clavulanateLevofloxacin 0.5 1 0.5 1 0.5 1 Moxifloxacin 0.25 0.25 0.25 0.25 0.250.25 Penicillin G 0.015 0.015 0.015 0.03 0.015 0.015

Against macrolide-susceptible strains, CEM-101 MICs were 0.008-0.03μg/ml and those against macrolide-resistant strains (all phenotypes)MICs were 0.015-1 μg/ml. TEL MICs were up to four-fold higher than thoseof CEM-101. The majority (17/19) of erm(B) strains were TEL-resistantwith MICs between 4 and 16 μg/ml while all had low CEM-101 MICs, similarto those of other resistance phenotypes (range 0.03-1 μg/ml). Results ofpneumococcal multistep resistance selection studies are presented in thefollowing Table.

S. pneumoniae multistep selection results. Strain No. Phenotype [R-Initial Selected Retest MIC after 10 Antibiotic-Strain determinates] MICResistance number Drug free Subcultures Drug (μg/ml) MIC # Pass CEM AZICLA TEL CLI 1077 Macrolide-S CEM-101 0.008 0.06 43 0.06 0.03 0.06 0.06a0.03 AZI 0.03 >64 29 0.03 >64 >64 0.125 4 CLR 0.016 0.008 50 — — — — —TEL 0.004 0.25 15 0.06 >64 >64 0.25 8 CLN 0.016 4 49 0.06 >64 >64 0.06 824 Macrolide- R[erm(B)] CEM-101 0.004 0.06 14 0.06 >64 >64 0.125 >64AZI >64 NT NT — — — — — CLR >64 NT NT — — — — — TEL 0.25 32 140.06 >64 >64 16 >64 CLN >64 NT NT — — — — — 3665 Macrolide- R [mef(A)]CEM-101 0.03 0.5 14 0.5 16 4 0.25 0.03 AZI 8 16 50 — — — — — CLR 2 16 260.06 8 8 0.06 0.03 TEL 0.125 2 14 0.03 4 2 0.25 0.03 CLN 0.016 0.03 50 —— — — — 1076 Macrolide-R [erm(B)&mef(A)] CEM-101 1 32 18 32 >64 >6432 >64 AZI >64 NT NT — — — — — CLR >64 NT NT — — — — — TEL 0.5 >64 142 >64 >64 >64 >64 CLN 64 NT NT — — — — — 1635 Macrolide- R [erm(A)]CEM-101 0.008 0.06 32 0.125 4 1 0.03 0.03 AZI 2 >64 14 0.004 >64 >640.004 0.03 CLR 0.5 >64 49 0.008 >64 >64 0.016 0.25 TEL 0.004 0.008 50 —— — — — CLN 0.06 >64 14 0.004 4 0.5 0.008 >64 2686 Macrolide- R [L4mutation] CEM-101 0.03 0.5 22 1 >64 32 0.25 0.03 AZI >64 NT NT — — — — —CLR 8 >64 14 0.03 >64 >64 0.06 0.03 TEL 0.06 0.5 25 0.016 >64 16 0.50.03 CLN 0.03 0.125 50 — — — — — 7127 Macrolide-S [S20N in L4, A105V inL22] CEM-101 0.008 0.125 16 0.06 0.06 0.125 0.06 0.03 AZI 0.06 0.5 290.016 1 0.5 0.016 0.06 CLR 0.03 16 15 0.008 >64 16 0.008 1 TEL 0.0080.06 38 0.008 0.06 0.06 0.03 0.03 CLN 0.03 0.25 43 0.004 0.03 0.0160.008 0.25 3009 Macrolide- R [23SrRNA mutation] CEM-101 0.016 0.25 200.25 >64 >64 0.06 1 AZI >64 NT NT — — — — — CLR 16 >64 25 0.03 >64 640.06 1 TEL 0.016 0.03 50 — — — — — CLN 1 2 50 — — — — — bCross-reactivity denoted in bold.

For pneumococci, parental MICs (μg/ml) were: CEM-101, 0.004-1; AZI,0.03-8; CLR, 0.016-16; TEL, 0.004-0.5; CLN, 0.016-1. Four strains withAZI, two with CLR, and two with CLN MICs≧64 μg/ml were not tested.CEM-101 MICs increased after 14-43 days in all 8 strains tested. For 7strains, MICs rose from 0.004-0.03 μg/ml (parents) to 0.06-0.5 μg/ml(resistant clones) in 14-43 days. For the eighth strain, containingerm(B)+mef(A), MICs rose from 1 μg/ml (parent) to 32 μg/ml (resistantclone) in 18 days. This CEM-101 resistant clone was subjected tosequencing analysis, which revealed no alterations in L4, L22 proteinsand II and V domain of 23S rRNA compared to parental sequences. AZI hadresistant clones after 14-29 days in 3/4 strains with MICs rising from0.03-2 μg/ml (parents) to 0.5->64 μg/ml (resistant clones). CLR hadresistant clones after 14-49 days in 5/6 strains with MICs rising from0.03-16 μg/ml (parents) to 16->64 μg/ml (resistant clones). TEL hadstable resistant clones after 14-38 days in 5/8 tested with MICs risingfrom 0.004-0.5 μg/ml (parents) to 0.06->64 μg/ml (resistant clones). CLNhad resistant clones after 14-43 days in 2/5 strains with MICs risingfrom 0.03-0.06 μg/ml (parents) to 0.25->64 μg/ml (resistant clones).

Results for S. pyogenes are shown in the following Table.

S. pyogenes multistep selection results Strain number Phenotype InitialRetest MIC after [Rdeterminates] MIC Selected 10 AntibioticfreeAntibiotic (μg/ml) Resistance Subcultures 2132 Macrolide-S CEM-101 0.0080.016 50 CEM AZI CLA TEL CLI AZI 0.06 1 28 0.016 1 0.25b 0.03 0.03 CLR0.03 0.016 50 — — — — — TEL 0.008 0.03 50 — — — — — CLN 0.06 0.06 50 — —— — — 2368 Macrolide-R [erm(B)] CEM-101 1 8 18 8 >64 >64 >64 >64 AZI >64NT NT — — — — — CLR >64 NT NT — — — — — TEL 8 >64 6 0.5 >64 >64 >64 >64CLN >64 NT NT — — — — — 2094 Macrolide-R [erm(A)] CEM-101 0.03 0.25 430.25 4 8 0.5 0.06 AZI 4 >64 5 0.016 >64 1 0.03 0.06 CLR 0.5 >64 60.016 >64 >64 0.03 0.06 TEL 0.03 0.25 22 0.03 >64 8 0.125 >64 CLN0.06 >64 34 0.03 16 1 0.03 >64 2011 Macrolide-R [mef(A)] CEM-101 0.1250.125 50 — — — — — AZI 4 32 35 0.06 16 4 0.25 0.06 CLR 4 8 50 — — — — —TEL 0.5 1 50 — — — — — CLN 0.06 0.06 50 — — — — — 237 Macrolide-R [LAmutation] CEM-101 0.03 0.25 20 0.5 4 1 1 0.03 AZI 4 8 50 — — — — — CLR0.25 1 50 — — — — — TEL 0.06 0.125 50 — — — — — CLN 0.06 0.5 43 0.03 80.5 0.06 1 bCross-reactivity denoted in bold.

Parental MICs (μg/ml) were: CEM-101, 0.008-1; AZI, 0.06-4; CLR, 0.03-4;TEL, 0.008-8; CLN 0.06. One strain with AZI, CLR and CLN MICs>64 μg/mlwas not tested. CEM-101 MICs increased after 18-43 days in 3/5 strains,rising from 0.03-1 μg/ml (parents) to 0.25-8 μg/ml (resistant clones).The resistant clone with a CEM-101 MIC of 8 μg/ml was subjected tosequencing analysis, which showed no changes in all genes (L4, L22 andII and V domain of 23S rRNA) tested. CEM-101 MICs for the remaining 2clones did not go above 0.25 μg/ml when passages were continued for themaximum 50 days. AZI had resistant clones after 5-35 days in 3/4 strainstested, with MICs rising from 0.06-4 μg/ml (parents) to 1->64 μg/ml(resistant clones). CLR had resistant clones after 6 days in 1/4 strainstested, with MICs rising from 0.5 μg/ml (parent) to >64 μg/ml (resistantclone). TEL had resistant clones after 6-22 days in 2/5 strains tested,with MICs rising from 0.03-8 μg/ml (parents) to 0.25->64 μg/ml(resistant clones). CLN had resistant clones after 34-43 days in 2/4strains tested with MICs rising from 0.06 μg/ml (parents) to 0.5->64μg/ml (resistant clones). Results of single step resistance selectionstudies for pneumococci are presented in the following Table.

S. pneumoniae single step mutation frequencies Strain Phenotype [R-number determinates.] Selecting Drug 2x MIC 4xMIC 8xMIC 1077Macrolide-S^(a) CEM-101 <9.1 × 10⁻⁹ <9.1 × 10⁻⁹ <9.1 × 10⁻⁹ Azithromycin 7.2 × 10⁻⁹  <1.9 × 10⁻¹⁰  <1.9 × 10⁻¹⁰ Clarithromycin  1.1 × 10⁻⁹  <1.2× 10⁻¹⁰  <1.2 × 10⁻¹⁰ Telithromycin  1.1 × 10⁻⁹  <5.5 × 10⁻¹⁰  <5.5 ×10⁻¹⁰ Clindamycin  <3.8 × 10⁻¹⁰  <3.8 × 10⁻¹⁰  <3.8 × 10⁻¹⁰ 24Macrolide-R^(a) CEM-101  1.8 × 10⁻⁷ <5.0 × 10⁻⁹ <5.0 × 10⁻⁹ [erm(B)]Azithromycin NT NT NT Clarithromycin NT NT NT Telithromycin  1.3 × 10⁻⁴ 2.5 × 10⁻⁶  1.7 × 10⁻⁶ Clindamycin NT NT NT 3665 Macrolide-R CEM-101 6.8 × 10⁻⁷  1.4 × 10⁻⁷  <4.5 × 10⁻¹⁰ [mef(A)] Azithromycin NT NT NTClarithromycin  5.0 × 10⁻⁷  <5.0 × 10⁻¹⁰  <5.0 × 10⁻¹⁰ Telithromycin 7.5 × 10⁻⁹  2.5 × 10⁻⁹  <2.5 × 10⁻¹⁰ Clindamycin  <2.4 × 10⁻¹⁰  <2.4 ×10⁻¹⁰  <2.4 × 10⁻¹⁰ 1076 Macrolide-R CEM-101 <2.5 × 10⁻⁸  7.5 × 10⁻⁹ 2.0 × 10⁻⁹ [erm(B)&mef(A)] Azithromycin NT NT NT Clarithromycin NT NTNT Telithromycin  6.5 × 10⁻⁶  9.7 × 10⁻⁶  4.8 × 10⁻⁶ Clindamycin NT NTNT 1635 Macrolide-R CEM-101  1.6 × 10⁻⁸  <2.0 × 10⁻¹⁰  <2.0 × 10⁻¹⁰[erm(A)] Azithromycin  <2.0 × 10⁻¹⁰  <2.0 × 10⁻¹⁰  <2.0 × 10⁻¹⁰Clarithromycin <3.1 × 10⁻⁹ <3.1 × 10⁻⁹ <3.1 × 10⁻⁹ Telithromycin <2.7 ×10⁻⁹ <2.7 × 10⁻⁹ <2.7 × 10⁻⁹ Clindamycin  7.3 × 10⁻⁷  5.5 × 10⁻⁷  5.6 ×10⁻⁷ 2686 Macrolide-R CEM-101 <2.5 × 10⁻⁹ <2.5 × 10⁻⁹ <2.5 × 10⁻⁹ [L4mutation] Azithromycin NT NT NT Clarithromycin NT NT NT Telithromycin 2.2 × 10⁻⁵  2.2 × 10⁻⁹ <1.1 × 10⁻⁹ Clindamycin <1.2 × 10⁻⁹ <1.2 × 10⁻⁹<1.2 × 10⁻⁹ 7127 Macrolide-S CEM-101  <2.0 × 10⁻¹⁰  <2.0 × 10⁻¹⁰  <2.0 ×10⁻¹⁰ [S20N in L4, Azithromycin  <2.0 × 10⁻¹⁰  <2.0 × 10⁻¹⁰  <2.0 ×10⁻¹⁰ A105V in Clarithromycin <1.0 × 10⁻⁹ <1.0 × 10⁻⁹ <1.0 × 10⁻⁹ L22]Telithromycin <5.0 × 10⁻⁹ <5.0 × 10⁻⁹ <5.0 × 10⁻⁹ Clindamycin <2.9 ×10⁻⁸ <2.9 × 10⁻⁸ <2.9 × 10⁻⁸ 3009 Macrolide-R CEM-101 <5.9 × 10⁻⁹ <5.9 ×10⁻⁹ <5.9 × 10⁻⁹ [23S rRNA Azithromycin NT NT NT mutation]Clarithromycin NT NT NT Telithromycin  3.8 × 10⁻⁷  <1.5 × 10⁻¹⁰  <1.5 ×10⁻¹⁰ Clindamycin  1.7 × 10⁻⁴  7.3 × 10⁻⁹  <1.2 × 10⁻¹⁰ ^(a)S =Susceptible; R = Resistant

The same 4 comparators used in multistep selection were tested for theirpropensity to produce spontaneous mutations. Mutant selectionfrequencies for CEM-101 ranged from <2.0×10⁻¹⁰-6.8×10⁻⁷ at 2×MIC to<2.0×10⁻¹⁰-9.1×10⁻⁹ at 8×MIC. These comparators had higher frequenciesof resistance: TEL, 1.1×10⁻⁹-1.3×10⁻⁴ at 2×MIC to <1.5×10⁻¹⁰-4.8×10⁻⁶ at8×MIC; CLN, <2.4×10⁻¹⁰-1.7×10⁻⁴ at 2×MIC to <1.2×10⁻¹⁰-5.6×10⁻⁷ at8×MIC; and CLR, <1.0×10⁻⁹-5.0×10⁻⁷ at 2×MIC to <1.2×10⁻¹⁰-<3.1×10⁻⁹ at8×MIC. A small number, 3 strains, were tested with AZI; mutant selectionfrequencies were <2.0×10⁻¹⁰-7.2×10⁻⁹ at 2×MIC to <1.9×10⁻¹⁰-<2.0×10⁻¹⁰at 8×MIC. Results of single step resistance selection studies for S.pyogenes are presented in the following Table.

S. pyogenes single step mutation frequencies Strain Phenotype [R- numberdeterminates.] Selecting Drug 2x MIC 4xMIC 8xMIC 2132 Macrolide-S^(a)CEM-101 <1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰ Azithromycin <1.0 × 10⁻¹⁰<1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰ Clarithromycin <1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰ <1.0× 10⁻¹⁰ Telithromycin <8.3 × 10⁻¹¹ <8.3 × 10⁻¹¹ <8.3 × 10⁻¹¹ Clindamycin<7.7 × 10⁻¹¹ <7.7 × 10⁻¹¹ <7.7 × 10⁻¹¹ 2368 Macrolide-R^(a) CEM-101 <5.9× 10⁻¹¹ <5.9 × 10⁻¹¹ <5.9 × 10⁻¹¹ [erm(B)] Azithromycin NT NT NTClarithromycin NT NT NT Telithromycin NT NT NT Clindamycin NT NT NT 2094Macrolide-R CEM-101  5.3 × 10⁻⁸  2.1 × 10⁻⁹  5.3 × 10⁻¹⁰ [erm(A)]Azithromycin NT NT NT Clarithromycin  1.7 × 10⁻⁷  1.0 × 10⁻⁷  5.0 × 10⁻⁹Telithromycin  7.7 × 10⁻⁸ <1.5 × 10⁻¹⁰ <1.5 × 10⁻¹⁰ Clindamycin  2.1 ×10⁻⁷  1.3 × 10⁻⁷  1.1 × 10⁻⁷ 2011 Macrolide-R CEM-101  3.9 × 10⁻⁸ <1.1 ×10⁻¹⁰ <1.1 × 10⁻¹⁰ [mef(A)] Azithromycin NT NT NT Clarithromycin NT NTNT Telithromycin  3.8 × 10⁻⁸ <6.3 × 10⁻¹⁰ <6.3 × 10⁻¹⁰ Clindamycin <1.0× 10⁻¹⁰ <1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰ 237 Macrolide-R CEM-101 <1.3 × 10⁻¹⁰<1.3 × 10⁻¹⁰ <1.3 × 10⁻¹⁰ [L4 mutation] Azithromycin NT NT NTClarithromycin <3.3 × 10⁻¹⁰ <3.3 × 10⁻¹⁰ <3.3 × 10⁻¹⁰ Telithromycin <2.0× 10⁻¹⁰ <2.0 × 10⁻¹⁰ <2.0 × 10⁻¹⁰ Clindamycin <1.0 × 10⁻¹⁰ <1.0 × 10⁻¹⁰<1.0 × 10⁻¹⁰ ^(a)S = Susceptible; R = Resistant

As with the pneumococci, the 4 comparators used in multistep selectionwere tested for their propensity to produce spontaneous mutations.Mutant selection frequencies for CEM-101 ranged from <5.9×10⁻¹¹-5.3×10⁻⁸at 2×MIC to <5.9×10⁻¹¹-<5.3×10⁻¹⁰ at 8×MIC. The following comparatorshad higher frequencies of resistance than CEM-101: CLN,<7.7×10⁻¹¹-2.1×10⁻⁷ at 2×MIC to <7.7×10⁻¹¹-1.1×10⁻⁷ at 8×MIC; and CLR,<1.0×10⁻¹⁰-1.7×10⁻⁷ at 2×MIC to <1.0×10⁻¹⁰-5.0×10⁻⁹ at 8×MIC. TELfrequencies were similar to CEM-101: <8.3×10⁻¹¹-7.7×10⁻⁸ at 2×MIC to<8.3×10⁻¹¹-<6.3×10⁻¹⁰ at 8×MIC. The mutation frequency for the onemacrolide-sensitive strain tested with AZI was <1.0×10⁻¹⁰ at 2× and8×MIC.

The compounds described herein demonstrate enhanced potency compared toTEL, with activity against TEL-intermediate and TEL resistant organisms.The compounds described herein show significantly greater potencyagainst phagocytized S. aureus when compared to TEL, AZI, and CLR. Thecompounds described herein are also about 50-fold and 100-fold morepotent than AZI against phagocytized L. monocytogenes and L.pneumophila. The compounds described herein exhibit the widest spectrumof activity against respiratory tract pathogens, including multidrug-resistant pneumococcus type 19A, compared to AZI, CLR,erythromycin, TEL, CLN, and quinupristin/dalfopristin. The compoundsdescribed herein are also potent against C. trachomatis, C. pneumoniae,human mycoplasmas and ureaplasmas, and the MICs also point to clinicalutility against most enterococci, gonococci, and Gram positiveanaerobes. The compounds described herein are active against commonorganisms that cause gastroenteritis, such as Campylobacter jejuni,Salmonella and Shigella, and is also active against Helicobacter pylori.The compounds described herein are shown to be more bactericidal againstseveral gram-positive species than TEL, with post-antibiotic effects of2.3-6.1 and 3.7-5.3 against gram-positive and -negative strains,respectively. The compounds described herein demonstrate significant invivo activity in a variety of murine infection models. Preliminarymultistep studies show that the compounds described herein have no oronly low variation in MICs in one strain each of S. aureus, Enterococcusfaecalis, and 2 S. pneumoniae; low rates of spontaneous mutants arefound in single step experiments.

The compounds described herein have MICs that are generally at least 1or 2 dilutions lower than those of TEL against all resistance phenotypesof S. pneumoniae and S. pyogenes tested, including drug-resistantpneumococcus type 19A and erm(B) positive S. pyogenes. CEM-101 yieldedclones with higher MICs in all 8 pneumococcal strains, but 7 of the 8strains have clones with CEM-101 MICs≦0.5 μg/ml and in only 1erm(B)+mef(A) strain with a parental MIC of 1 μg/ml was a resistantclone found with an MIC of 32 μg/ml. In 2 of the 3 resistant S. pyogenesCEM-101 clones [parents erm(A), L4] MICs were 0.25 μg/ml and only in the1 strain with erm(B) did CEM-101 MICs rise from 1 to 8 μg/ml. Singlestep studies also showed low yields of spontaneous mutations compared toother agents tested.

Based on pharmacokinetics reported from Phase 1 clinical trialsrecommendations for tentative CEM-101 susceptibility breakpoints havebeen set at ≦1 μg/ml as susceptible and ≧4 μg/ml as resistant againststreptococci.

Example.

Staphylococci, β-Haemolytic and Viridans Group Streptococci. Acollection of 2006-2007 clinical isolates were S tested by CLSI methods(M7-A7) with associated interpretive criteria (M100-S18) and supplements(2-5% LHB) for streptococcal tests. CEM-101, TEL (TEL) and 10comparators were used versus 201 S. aureus (75 WT-MRSA, 75 WT-MSSA, 30CA-MRSA, 17 VISA or hVISA, 7 VRSA), 100 coagulase-negative staphylococci(CoNS; 10 species), 100β-haemolytic (BHS; 30 group A, 31 group B, 14group C, 9 group F, 16 group G) and 51 viridans group streptococci (VGS;5 species), see Table.

MSSA strains were slightly more CEM-101-S (MIC50, 0.06 μg/ml) that MRSAor CA-MRSA strains (MIC50, 0.12 μg/ml). VISA, hVISA and VRSA weregenerally more refractory to CEM-101 and TEL. CEM-101 was 2-fold morepotent than TEL against all staphylococci. Streptococci were very S toCEM-101 (MIC90, 0.03-0.06 μg/ml) and TEL was 4-fold less active withnon-S isolates of BHS observed. ERY-R staphylococci remained CEM-101-Sexcept for TEL- and CLN (CC)-R isolates, but all BHS and VGS were S toCEM-101.

CEM-101 MIC TEL MIC (μg/ml) (μg/ml) Organisms (no.) 50% 90% Range 50%90% Range MSSA (75) 0.06 0.12 0.03->16 0.12 0.25 0.06->16 MRSA (75)0.12 >16 0.03->16 0.25 >16 0.06->16 CA-MRSA (30) 0.12 0.12  0.06-0.120.25 0.25 0.12-0.5  VISA, hVISA (14) >16 >16 0.06->16 >16 >16 0.25->16VRSA (7) >16 — 0.12->16 >16 — 0.12->16 CoNS (100) 0.06 >16 0.03->160.12 >16 0.03->16 BHS (100) 0.015 0.03 ≦0.008-0.12    0.03 0.12≦0.008-2      VGS (51) ≦0.008 0.06 ≦0.008-0.12    0.015 0.25≦0.008-0.5   CEM-101 was potent against all staphylococci (MIC50, 0.06 μg/ml), exceptCC-R strains; and inhibited all streptococci at ≦0.12 μg/ml. Theactivity was greater than TEL by 2- to 4-fold.

1.-40. (canceled)
 41. A method for treating a disease in a host animal,where the disease is caused at least in part by one or more bacteriaresistant to azithromycin or clarithromycin, or both, the methodcomprising the step of administering an effective amount to the hostanimal of a compound of the formula

or a salt thereof, wherein: R₁₀ is hydrogen or acyl; W is H, F, Cl, Br,I, or OH; A is CH₂, C(O), C(O)O, C(O)NH, S(O)₂, S(O)₂NH, C(O)NHS(O)₂; Bis C₀-C₁₀ alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl, or C₄-C₁₀alkenylalkynyl; and C is hydrogen, hydroxy, acyl, acyloxy, sulfonyl,ureido, or carbamoyl, or alkyl, alkoxy, heteroalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl, each of which is optionally substituted.42. (canceled)